Endothelial cell-leukocyte adhesion molecules (ELAMs) and molecules involved in leukocyte adhesion (MILAs)

ABSTRACT

Antibodies specific for ELAMs are disclosed.

TECHNICAL FIELD OF INVENTION

This invention relates to molecules involved in the adhesion ofleukocytes to endothelial cells during inflammation and to DNA sequencesthat code on expression for them. More particularly, it relates toEndothelial Cell Adhesion Molecules (ELAMs), including ELAM1 andVascular Cell Adhesion Molecule 1 and 1b (VCAM1 and VCAM1b). It alsorelates to molecules on the surface of leukocytes involved in leukocyteadhesion to endothelial cells (MILAs). These include CDX, a moleculeinvolved in the ELAM1 adhesion pathway, and VLA4, the ligand of VCAM1and VCAM1b. This invention also relates to clone 7.2 and clone 1. TheseDNA sequences encode protein 7.2 and protein 1, respectively, which areinvolved in the expression of CDX. These proteins appear to be1,3-fucosyl transferases that glycosylate CDX. This invention alsorelates to Pseudo-X and Pseudo-X₂, proteins that appear on COS 7 and CHOcells, respectively, that have been transfected with clone 7.2. Cellsexpressing those proteins bind ELAM1 and are recognized by anti-CDXmonoclonal antibodies. This invention further relates to antibodies thatrecognize these adhesion molecules and anti-idiotype antibodies thatrecognize both those antibodies and the ligands or receptors for theadhesion molecules. The invention also relates to antisense DNA and RNAmolecules complementary to mRNA for such adhesion molecules and alsorelates to ribozymes which recognize mRNA for such molecules. Theinvention also relates to methods for using the aforementionedmolecules, DNA sequences, antibodies, anti-idiotype antibodies,antisense molecules and ribozymes, for example in developing diagnosticand therapeutic agents to detect or inhibit leukocyte adhesion toendothelial cells.

BACKGROUND OF THE INVENTION

Inflammation is the response of vascularized tissues to infection orinjury. Clinically it is accompanied by four classic signs: redness,heat, pain and swelling. Its course may be acute or chronic.

At the cellular level, inflammation involves the adhesion of leukocytes(white blood cells) to the endothelial wall of blood vessels and theirinfiltration into the surrounding tissues. (Harlan, 1985.) Acuteinflammation is characterized by the adhesion and infiltration ofpolymorphonuclear leukocytes (PMNs). (Harlan, 1987 and Malech andGallin, 1987.) PMN accumulation in the tissues reaches its peak betweentwo and one half to four hours after an inflammatory stimulus and ceasesby about twenty-eight hours. (Bevilacqua and Gimbrone, 1987.) Incontrast, chronic inflammation is characterized by the adhesion andinfiltration of other leukocytes, especially monocytes and lymphocytes.

In normal inflammation, the infiltrating leukocytes phagocytize invadingorganisms or dead cells, and play a role in tissue repair and the immuneresponse. However, in pathologic inflammation, infiltrating leukocytescan cause serious and sometimes deadly damage. Rheumatoid arthritis andatherosclerosis are examples of chronic inflammatory diseases in whichmononuclear leukocytes infiltrate the tissues and cause damage. (Houghand Sokoloff, 1985 and Ross, 1986.) Multiple organ failure syndrome,adult respiratory distress syndrome (ARDS), and ischemic reperfusioninjury are acute inflammations in which infiltrating PMNs cause thedamage (Harlan, 1987 and Malech and Gallin, 1987). In multiple organfailure syndrome, which can occur after shock such as that associatedwith severe burns, PMN-mediated damage exacerbates the injury. In ARDS,PMNs cause the lungs to fill with fluid, and the victim may drown. Inischemic reperfusion injury, which occurs when tissue cut off from thesupply of blood is suddenly perfused with blood (for example after heartattack, stroke, or limb re-attachment), PMN adhesion causes serioustissue damage (Harlan, 1987).

Recognizing that leukocyte infiltration is the cause of muchinflammation-related pathology and that leukocyte adhesion is the firststep in infiltration, investigators have recently focused attention onthe mechanism of leukocyte binding to the endothelial cell surface.Studies show that binding is mediated by cell-surface molecules on bothendothelial cells and leukocytes which act as receptor and ligand(Harlan et al., 1987; Dana et al., 1986; and Bevilacqua et al., 1987a).

During the course of inflammation, certain inflammatory agents can acton the leukocytes, making them hyperadhesive for endothelium. Knowninflammatory agents include leukotriene-B4 (LTB4), complement factor 5a(C5a), and formyl-methionyl-leucyl-phenylalanine (FMLP). These agentsactivate a group of proteins called LeuCAMs. The LeuCAMs are dimers ofthe CD11 and CD18 proteins. One of the LeuCAMs, CD11a/CD18 (also calledLFA1) binds to a receptor on endothelial cells called ICAM1(intercellular adhesion molecule 1). (Harlan, 1985 and Dana et al.,1986.) Investigators have shown that monoclonal antibodies (Moabs) toLeuCAMs inhibit PMN adhesion to endothelium both in vitro and in vivo.(Arfors, 1987; Vedder et al., 1988; and Todd, 1989.)

Other inflammatory agents act directly on endothelial cells tosubstantially augment leukocyte adhesion. These agents include thecytokines interleukin-1 (IL-1), lymphotoxin (LT) and tumor necrosisfactor (TNF), as well as the bacterial endotoxin, lipopolysaccharide(LPS). For example, IL-1 has been shown to stimulate adhesion of PMNs,monocytes, and the related cell lines HL-60 (PMN-like) and U937(monocyte-like), to human endothelial cell monolayers. The action isboth time-dependent and protein-synthesis dependent. (Bevilacqua et al.,1987a; Bevilacqua et al., 1987b; and Bevilacqua et al., 1985.)

Current evidence indicates that these agents induce a group of moleculeson the endothelial cell surface called ELAMs (endothelial cell-leukocyteadhesion molecules). To date investigators have identified two of thesemolecules, intercellular adhesion molecule 1 (ICAM1) and endothelialcell-leukocyte adhesion molecule 1 (ELAM1). (Simmons et al., 1988;Staunton et al., 1988; and Bevilacqua et al., 1987b.) ICAM1 is found onmany cell types, and its expression on vascular endothelium is stronglyupregulated both in vitro and in vivo by the inflammatory cytokinesinterleukin-1 (IL-1), tumor necrosis factor-α (TNF), and gammainterferon (IFN-γ). (Pober et al., 1986; Dustin and Springer, 1988; andCotran and Pober, 1988.)

ELAM1 was initially detected and characterized by a monoclonal antibodythat partially blocked PMN adhesion to cytokine-treated human umbilicalvein endothelial cells (HUVECs). ELAM1 is a 116 kD cell surfaceglycoprotein rapidly synthesized by HUVECs in response to theinflammatory cytokines IL-1 or TNF, but not IFN-γ. (Bevilacqua et al.,1987b.) Unlike ICAM1, ELAM1 appears to be expressed only in endothelium,and its expression is transient even in the continued presence ofcytokine. Like ICAM1, ELAM1 is present at inflammatory sites in vivo.Immunohistologic studies show that it exists at sites of acute, but notchronic, inflammation and is absent from the non-inflamed vessel wall.(Cotran et al., 1986 and Cotran and Pober, 1988.) Therefore, ELAM1appears to be a major mediator of PMN adhesion to the inflamed vascularwall in vivo. Importantly, the presence of ELAM1 on the cell surfacefollows the natural course of acute inflammation, appearing a few hoursafter stimulation and gradually dissipating within a day. (Bevilacqua etal., 1987b.)

Indirect evidence suggests that other ELAMs exist. Although inflammatoryagents induce binding of PMNs, monocytes, and lymphocytes to endotheliumin vitro, Moabs against ELAM1 inhibit only the binding of PMNs andrelated cells. (Bevilacqua and Gimbrone, 1987.) Furthermore, maximalaccumulation of lymphocytes and monocytes at sites of inflammation invivo occurs at about twenty-four hours, when ELAM1 expression hasreturned to basal levels. On the basis of such information investigatorsinferred the presence of other ELAMs that mediate binding of theselymphocytes and monocytes. (Bevilacqua et al., 1987b.) As set forth indetail below, we have characterized and cloned two more ELAMS,designated VCAM1 and VCAM1b, that mediate binding of lymphocytes toendothelial cells. ELAMs accordingly may be regarded as a family ofmolecules.

A growing body of evidence indicates that ELAMs may play important rolesin a wide range of pathological states involving cell-cell recognition,including tumor invasion, metastasis and viral infection. (Harlan, 1985;Wallis and Harlan, 1986; Bevilacqua et al., 1987a; and Cotran and Pober,1988.)

The adhesion of leukocytes to cells expressing ELAMs suggests theexistence on leukocytes of ELAM ligands. One such molecule is the ICAM1ligand, lymphocyte function associated antigen 1 (LFA1). LFA1 is one ofa trio of heterodimeric molecules known as the 82 integrins or theCD11/18 family. (Dustin et al., 1986; Rothlein et al., 1986; and Marlinand Springer, 1987.) Recent studies show that the ICAM1/LFA1 pathwayplays a role in both lymphocyte and polymorphonuclear leukocyte (PMN)adhesion to endothelial cells in vitro. (Dustin and Springer, 1988;Smith et al., 1989.) We report here the isolation of a molecule involvedin leukocyte adhesion to endothelial cells (MILA) which may prove to bean ELAM1 ligand. The molecule, designated CDX, is a protein ofapproximately 150 kD and was isolated from HL-60 cells. Monoclonalantibodies that recognize CDX inhibit the binding of PMNs and HL-60cells to ELAM1-expressing cells. Furthermore, CDX is present onleukocyte cell types known to adhere to ELAM1 and is absent fromleukocyte cell types and other cell types that do not adhere to ELAM1.Thus, CDX is a molecule expressed on certain leukocytes that plays animportant role in ELAM1-mediated leukocyte-endothelial cell adhesion. Wealso report the isolation and sequencing of cDNA encoding moleculesinvolved in CDX expression.

We also report the identification of a VCAM1 and VCAM1b ligand, VLA4.(Hemler and Takada, EP 330 506). Antibodies specific for the α⁴ and β₁subunits of VLA4 completely eliminate binding of VLA4-expressing cellsto VCAM1.

Because leukocyte adhesion to the vascular wall is the first step ininflammation, therapies directed to preventing this step are attractivefor the treatment of pathologic inflammation. Clinicians are alreadytesting, with some success, therapies based on inhibitingleukocyte-mediated adhesion. One such approach involves Moab binding tothe leukocyte cell-surface complex, CD11/CD18, to inhibit PMN adhesion.(Arfors et al., 1987; Vedder et al., 1988; and Todd et al., 1989.)

We believe that alternative therapies for preventing leukocyte adhesion,based on endothelial cell-mediated binding, and on ELAMs and MILAs(including ELAM ligands), in particular, are more promising. The ELAMsystem is particularly appealing for two reasons: First, because ELAMexpression on endothelial cells is induced rather than constitutive,ELAMs are concentrated at sites of inflammation and are limited innumber. This means that adhesion inhibitors need act only locally and,consequently, would be effective at lower doses than inhibitors directedto constitutively expressed molecules. Second, ELAM binding is selectivefor different leukocyte classes. For example, ELAM1 binds PMNs, andVCAM1 binds lymphocytes. Therefore, these therapies would be specificfor certain classes of leukocytes and would not affect the circulationor migration of other leukocyte classes. Furthermore, for the abovereasons, such therapies may prove to be cheaper and less toxic.

ELAM-based approaches to therapy require, as starting materials, bothELAMs and MILAs in highly purified form, free of normally associatedanimal proteins. There is also a need for methods to produce thesemolecules. These and other needs have now been met as described herein,by isolating DNA sequences that code on expression for particularadhesion molecules and by constructing recombinant DNA molecules andexpression vehicles for their production.

SUMMARY OF THE INVENTION

It is the principal object of this invention to provide new means tostudy, diagnose, prevent and treat inflammation. More particularly, itis an object of this invention to provide molecules involved inleukocyte binding to endothelial cells and to isolate other moleculeswhich are themselves useful in inhibiting the endothelial cell bindingof leukocytes.

This invention provides DNA sequences that code on expression forendothelial cell-leukocyte adhesion molecules (ELAMs), genomic DNAsequences for ELAMs (including ELAM expression control sequences),recombinant DNA molecules containing these DNA sequences, unicellularhosts transformed with these DNA molecules, processes for producingELAMs, and ELAM proteins essentially free of normally associated animalproteins. The present invention also provides for antibody preparationsreactive for ELAMs.

This invention also provides DNA sequences that code on expression formolecules involved in leukocyte adhesion to endothelial cells (MILAs).MILAs will include leukocyte surface molecules that bind directly toELAMs, i.e., ELAM ligands. Monoclonal antibodies recognizing ELAMligands can inhibit ELAM/ELAM ligand binding directly. MILAs will alsoinclude leukocyte surface molecules that are involved indirectly inadhesion, for example molecules that inhibit ELAM/ELAM ligand binding byinteracting with a third molecule, such as a monoclonal antibody. Suchmolecules may act, for example, by changing the surface conformation ofan ELAM ligand so that its affinity for the ELAM is reduced. Thisinvention also provides recombinant DNA molecules containing MILA DNAsequences and unicellular hosts transformed with them. It also providesfor MILA proteins essentially free of normally associated animalproteins, methods for producing MILAs, and monoclonal antibodies thatrecognize MILAs, particularly CDX.

This invention provides DNA sequences encoding molecules that causeseveral cell lines, including COS, CHO and R1.1, both express surfaceglycoproteins that are recognized by anti-CDX (α-CDX) antibodies and tobind to ELAM1. This invention provides, in particular, clone 7.2 andclone 1, and protein 7.2 and protein 1, respectively. These proteinsappear to be 1,3-fucosyl transferases.

This invention also provides the glycoproteins, Pseudo-X and Pseudo-X₂,which cause COS cells and CHO cells, respectively, to bind ELAM1 and tobe recognized by α-CDX antibodies.

This invention further provides methods for inhibiting PMN binding toendothelial cells involving the use of ELAMs, MILAs including ELAMligands, or portions of those molecules to block receptors or ligands.It also relates to the use of antisense nucleic acids and ribozymes toinhibit ELAM expression. The invention also relates to methods foridentifying binding inhibitors by screening molecules for their abilityto inhibit binding of an ELAM to its ligand. It provides methods foridentifying ELAMs and their ligands. One such method involves usinganti-idiotypic antibodies against antibodies that recognize ELAMs orELAM ligands.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the composite ELAM1 cDNA sequence and deduced amino acidsequence derived from the DNA sequences of ELAM pCDM8 clone 6, pSQ148and pSQ149. The nucleotides are numbered from 1 to 3863. Throughout thisapplication we refer to the coding DNA sequence of this figure as theDNA sequence for ELAM1. We also refer to the molecule comprising theamino acid sequence depicted in this figure as ELAM1.

FIG. 2 depicts the DNA sequence of the synthetic polylinker of pNN11.

FIG. 3 depicts the sequence of cDNA coding for VCAM1 and the deducedamino acid sequence of VCAM1 derived from AM pCDM8 clone 41. Thenucleotides are numbered 1 to 2811. In this application we refer to thecoding DNA sequence of this figure as the DNA sequence for VCAM1. Wealso refer to the molecule comprising the amino acid sequence depictedin this figure as VCAM1.

FIG. 4 depicts the sequence of cDNA coding for VCAM1b and the deducedamino acid sequence of VCAM1b derived from VCAM1b pCDM8 clone 1E11. Thenucleotides are numbered 1 to 3080. In this application we refer to thecoding DNA sequence of this figure as the DNA sequence for VCAM1b. Wealso refer to the molecule comprising the amino acid sequence depictedin this figure as VCAM1b.

FIG. 5 depicts the domain structure of VCAM1. The amino acids areindicated according to the one letter code used by the University ofWisconsin Genetics Computer Group. (Devereux et al., 1984.)

FIG. 6 depicts the domain structure of VCAM1b. The amino acids areindicated according to the one letter code used by the University ofWisconsin Genetics Computer Group. (Devereux et al., 1984.)

FIG. 7 depicts the DNA sequence of portions of the 5′ untranslated anduntranscribed region of ELAM1 derived from clone EL1-07.

FIG. 8 depicts the DNA sequence of portions of the 5′ untranslated anduntranscribed region of VCAM1 derived from clone VC1-16.

FIG. 9 depicts the sequence of cDNA coding for protein 7.2 and thededuced amino acid sequence of protein 7.2 derived from pSQ219 and CDXpCDM8 clone 7.2. The nucleotides are numbered 1-2175. In thisapplication we refer to the coding DNA sequence of this figure as theDNA sequence for clone 7.2. We also refer to the polypeptide comprisingthe amino acid sequence depicted in this figure as protein 7.2.

FIG. 10 depicts the sequence of cDNA coding for protein 1 derived fromclone 1. The nucleotides are numbered 1-2861. In this application werefer to the coding DNA sequence of this figure as the DNA sequence forclone 1. We also refer to the polypeptide comprising the amino acidsequence depicted in this figure as protein 1.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with this detailed description, the following definitionsapply:

Expression control sequence—A DNA sequence that controls and regulatesthe transcription and translation of another DNA sequence.

Operatively linked—A DNA sequence is operatively linked to an expressioncontrol sequence when the expression control sequence controls andregulates the transcription and translation of that DNA sequence. Theterm “operatively linked” includes having an appropriate start signal(e.g., ATG) in front of the DNA sequence to be expressed and maintainingthe correct reading frame to permit expression of the DNA sequence underthe control of the expression control sequence and production of thedesired product encoded by the DNA sequence. If a gene that one desiresto insert into a recombinant DNA molecule does not contain anappropriate start signal, such a start signal can be inserted in frontof the gene.

Antibody—An immunoglobulin molecule or functional fragment thereof, suchas Fab, F(ab′)₂ or dAb. An antibody preparation is reactive for aparticular antigen when at least a portion of the individualimmunoglobulin molecules in the preparation recognize (i.e., bind to)the antigen. An antibody preparation is non-reactive for an antigen whenbinding of the individual immunoglobulin molecules in the preparation tothe antigen is not detectable by commonly used methods.

Standard hybridization conditions—salt and temperature conditionssubstantially equivalent to 5×SSC and 65° C. for both hybridization andwash. Under standard hybridization conditions the DNA sequences of thisinvention will hybridize to other DNA sequences having sufficienthomology, including homologous sequences from different species. It isunderstood that the stringency of hybridization conditions is a factorin the degree of homology required for hybridization.

DNA sequences—The DNA sequences of this invention refer to DNA sequencesprepared or isolated using recombinant DNA techniques. These includecDNA sequences, DNA sequences isolated from their native genome, andsynthetic DNA sequences. The term as used in the claims is not intendedto include naturally occurring DNA sequences as they exist in Nature.

ELAM—A molecule expressed on the surface of endothelial cells thatmediates adhesion of leukocytes to endothelial cells.

MILA—A molecule expressed on the surface of leukocytes that is involvedin ELAM-mediated binding to endothelial cells. This includes ELAMligands, i.e., molecules that bind directly to ELAMs.

As described below, we have isolated and sequenced cDNAs from ELAMmRNAs, expressed ELAM molecules in an appropriate host, isolated andsequenced cDNAs encoding MILAs, and isolated and expressed DNA sequencesfor MILAs.

Expression of recombinant DNA molecules according to this invention mayinvolve post-translational modification of a resultant polypeptide bythe host cell. For example, in mammalian cells expression might include,among other things, glycosylation, lipidation or phosphorylation of apolypeptide, or cleavage of a signal sequence to produce a “mature”protein. Accordingly, as used herein, the term “protein”, includingELAM, MILA, protein 1, protein 7.2, Pseudo-X and Pseudo-X₂ encompassfull-length polypeptides and modifications or derivatives thereof, suchas glycosylated versions of such polypeptides, mature proteins,polypeptides retaining a signal peptide, truncated polypeptides havingcomparable biological activity, and the like.

ELAMs are expressed on the surface of endothelial cells only duringinflammation. We utilized this phenomenon to isolate ELAM cDNAs. We havedesignated the polypeptides encoded by our cDNA isolates ELAM1, VCAM1and VCAM1b. The first step involved in the isolation was selection ofcells that differentially expressed the ELAM molecules. We chose humanumbilical vein endothelial cells because they produce ELAMs when inducedby the inflammatory cytokine, IL-1β. However, the practitioner is notlimited to this cytokine, to this cell type, or even to human cells inparticular. Other mammalian cells, e.g., baboon endothelial cells, arealso known to produce ELAMs. (Cotran and Pober, 1988.)

The next step was to isolate mRNA from cells expressing ELAMS, in thiscase, IL-1β-induced HUVECs, and to create a cDNA library from them. Manymethods are known for isolating mRNA and for producing cDNA from it.(See, e.g., Gubler and Hoffman, 1983 and Maniatis et al., 1982.)

We then inserted the cDNA into an appropriate vector. We chose theeukaryotic expression vector pCDM8, described by Brian Seed. (Seed,1987.) This plasmid has several advantages including a high copy numberin E. coli, a eukaryotic promoter, and high level of expression intransient expression systems such as COS 7 cells. However, several othervector systems are available. (See, e.g., Cate et al., 1986.)

After constructing a cDNA library, the next step was to isolate from itclones containing ELAM cDNA sequences. There are currently many ways toisolate cDNA for a differentially expressed mRNA. These include, forexample, (1) plus/minus screening with labeled cDNA; (2) production ofsubtracted cDNA libraries; and (3) screening with subtractive cDNAprobes. (Davis, 1986; Sargent, 1987; Davis et al., 1985, Hedrick et al.,1984; and Duguid et al., 1988.) We chose the third technique, screeningwith subtractive cDNA probes, and produced a cDNA sublibrary enrichedfor ELAM sequences.

As we will describe in more detail below, we produced a subtractive cDNAprobe enriched for mRNA produced by cytokine-induced, but not uninducedcells. Then we probed the cytokine-induced cDNA library with thesubtracted cDNA probe using techniques well known to the art. Thisenabled us to isolate clones forming a sublibrary enriched for ELAMsequences.

At this point we used two techniques to identify clones that containedcDNA for ELAM sequences. In a first method, we tested clones forexpression of ELAM activity in an appropriate eukaryotic expressionsystem. One can use a variety of direct expression techniques, includingantibody screening of fusion proteins encoded by cDNA cloned in λGT11(Young and Davis, 1983; Young and Davis, 1984); or activity assay ofoocyte-conditioned media after injection of mRNA from cloned cDNA, orfrom plasmid or phage DNA carrying SP6/T7 promoters. Alternatively, onecan make libraries in plasmid, phage, and cosmid vectors containing avariety of promoter, selection and replication elements. Animal cellsmay be transfected with the library for transient or stable expression.Transfection can be accomplished by a variety of methods. For transientexpression, investigators have used spheroplast fusion, DEAE dextran,and electroporation. For stable expression they have used calciumphosphate, spheroplast fusion, and electroporation. We used COS 7 cells,a transient expression system, transfected by spheroplast fusion.(Aruffo and Seed, 1987.)

Until recently, identification of cloned molecules by direct expressionhas required sensitive assays and has been restricted to lymphokines.However, cDNA cloning of single-chain cell-surface molecules inefficient transient expression vectors (see, e.g., Seed and Aruffo, 1987and Seed, 1987), either by antibody “panning” technology (Wysocki andSato, 1978) or by identification of functional molecules by FACS(Yamasaki et al., 1988), has expanded the range of cloned molecules thatone can identify by direct expression. We have extended this technologyby using an adhesion assay in that an appropriate cell type, expressingthe ligand for the cloned molecule, is used to identify that molecule.

We detected ELAM expression by testing the ability of transfected cellsto bind either the human neutrophil-like cell line, HL-60 (Bevilacqua etal., 1985), or the human B-lymphocyte-like cell line, RAMOS (AmericanType Culture Collection, ATCC accession no. CRL 1596, human Burkittlymphoma). We describe this in more detail below. Because thetransfected cells were non-human, those producing human ELAMpolypeptides did so in substantially purified form and essentially freeof normally associated animal proteins. We picked cells that testedpositive in this assay, collected the plasmid DNA, and isolated theinserts from them. These inserts contained DNA sequences encoding ELAM1(selected by adhesion to HL-60 cells) and VCAM1 (selected by adhesion toRAMOS cells).

In a second method, we identified cDNA inserts from the enrichedsublibrary that hybridized on a Northern blot to a 4 kb band of induced,but not uninduced, mRNA. Two of these inserts contained DNA sequencesfor ELAM1. Other inserts from the sublibrary encode different inducedmRNAs.

We isolated a cDNA for another VCAM, VCAM1b, by probing theIL-1β-induced HUVEC cDNA library with a random-primed oligonucleotide³²P-labeled probe derived from the VCAM1 cDNA sequence. VCAM1b is largerthan VCAM1.

Using the clones identified by these three methods, we determined thesequences of cDNAs for ELAM1 and VCAM1 and 1b. It should be noted thatdue to the degeneracy of the genetic code, one may alter many of thenucleotides of these sequences and retain DNA sequences that code onexpression for an amino acid sequence identical to those encoded by theDNA sequences we have presented in FIGS. 1, 3 and 4. Additionally, DNAsequences for fragments of the ELAM cDNA sequences, or DNA sequencesthat are substantially homologous to the ELAM cDNA sequences and thatthemselves encode ELAM polypeptides, would hybridize to the disclosedELAM cDNA sequence under standard hybridization conditions.

From the DNA sequences described above, we deduced the amino acidsequences of ELAM1, VCAM1 and VCAM1b. It should be clear that given thecurrent state of the protein-engineering art, an artisan could makepurposeful alterations, insertions or deletions in these amino acidsequences and obtain a variety of molecules having substantially thesame biological or immunological activities as those of the molecules wehave disclosed herein.

We have also isolated genomic DNA sequences, including transcriptionalpromoters, for the ELAM1 and VCAM1 and 1b genes. We screened a humangenomic library with ³²P-labeled probes derived from the coding regionsof the ELAM1 or VCAM1 DNA sequences. This yielded clones that containedportions of the 5′ untranscribed and untranslated regions of both theELAM1 and VCAM1 gene.

ELAM1 and VCAM1 transcriptional promoters have a number of uses. First,they are useful to construct vectors inducible by cytokines (such as TNFor IL-1), and bacterial lipopolysaccharide (LPS), or any other agentfound to induce expression of ELAMs in endothelial cells. Such vectorsmay be useful, for example, in gene transfer assays, wherein theinducible promoter is positioned so that it drives transcription of areporter gene such as chloramphenicol acetyltransferase,beta-galactosidase, luciferase, etc. This construct will then beintroduced transiently or stably into an appropriate mammalian cellline. Potential inhibitors or stimulators of induction can then beassayed by measuring their effect on induction by any or all of theinducers listed above.

We have also isolated a hybridoma producing monoclonal antibodiesrecognizing ELAM1, designated BB11. We describe its production inExample V, infra.

VCAM1 is involved in T and B cell binding to endothelial cells. T cellsactivated by lectin stimulation or by a specific antigen bind to HUVECsin vitro. This binding is mediated in part by the ICAM/LFA1 pathway,since monoclonal antibodies that bind to an inhibitory epitope on CD18(the common B chain of LFA1) partially inhibit T cell binding. We foundthat anti-CD18 and anti-VCAM1 monoclonals completely inhibited binding.Coupled with the observations that humans deficient in CD18 exhibitnormal recruitment of lymphocytes to sites of inflammation, and thatactivated T cells do not recirculate through the lymphatic system (i.e.,they will not exit from the blood stream except at sites ofinflammation), this implies that VCAM1 is central to activated T cellmigration in vivo. Thus, VCAM1 serves to focus all activated T cellsinto an inflammatory site. Since the presence of activated T cells isthe hallmark of numerous inflammatory and autoimmune diseases, this inturn implies that inappropriate expression of VCAM1 might be thefundamental pathochemical characteristic of such diseases. Therefore,the VCAM1 pathway may provide a key intervention point for diseaseswhere activated T cell recruitment is involved, e.g., arthritis, lupus,multiple sclerosis, etc. Therefore, we disclose a therapeutic treatmentto inhibit T cell binding to the endothelium by blocking the VCAM1binding pathway. This may be accomplished by any of the means wedescribe herein.

The DNA sequence of VCAM1 reveals that the molecule has no structuralsimilarity to ELAM1 but is a member of the immunoglobulin supergenefamily. Three of the Ig superfamily members are established cell-celladhesion molecules. These are NCAM, CEA, and ICAM1. NCAM binds to itselfon the surface of other cells (homotypic adhesion) thus promotingadhesion between cells of the same type. The function of CEA was unknownuntil recently, when it was discovered to function as an adhesionmolecule, mediating homotypic aggregation of colon tumor cells as wellas cells transfected with the cDNA for CEA. (Benchimol et al., 1989.)ICAM1 is a ligand for the leukocyte surface protein, LFA1, and mediatesboth leukocyte-leukocyte and leukocyte-endothelial cell adhesion.(Staunton et al., 1988.) ICAM1 and VCAM1 possess some functionalsimilarities, e.g., both are induced in endothelial cells aftertreatment with cytokines, and both mediate adhesion of lymphocytes andrelated cell lines. The ligand for ICAM1, LFA-1, has beenwell-characterized. The ligand for VCAM1 has been identified as VLA4(see, infra). ICAM1 is believed to play a role not only in the migrationof lymphocytes to sites of inflammation in vivo but also in numerouslymphocyte functions related to the immune response. Additionally, ICAM1has recently been shown to be the receptor for many of the rhinoviruses.Receptors for other viruses (e.g., polio, HIV) are also members of theIg superfamily. (White and Littman, 1989.) Thus, VCAM1 may play acritical role in both immune regulation and viral infection.

Both CEA and ICAM1 are expressed on tumor cells. CEA has been used as adiagnostic marker for colon cancer for many years. Recent diagnostictechniques include the use of radioimmunoconjugates. in which anti-CEAantibodies are bound to radioactive markers and introduced into thepatient. Using this method, clinicians have been able to identify tumorsas small as three millimeters. (Goldenberg, 1989.)

Investigators are also exploring radioimmunotherapy and immunotoxintherapy. Radioimmunotherapy involves the use of radioimmunoconjugates inwhich nuclides such as ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re and the like are bound toantibodies recognizing a particular surface antigen. Immunotoxins areantibodies conjugated with cell toxins, such as Pseudomonas exotoxin andthe like. Upon injection, these conjugated antibodies target the toxicagents to cells expressing the antigen. In accordance with thisinvention, radioactive markers, nuclides and cellular toxins may beconjugated with VCAM1 and 1b or antibodies recognizing them to targetcells expressing VCAM1 ligands (e.g., VLA4) or VCAM1.

The discovery of new ELAMs or the future discovery of ELAMs or MILAsbeing expressed on other cells, such a tumor cells, also makes possiblenew TIL therapies. For example, where a tumor is discovered whichexpresses an ELAM on its surface, the tumor can be biopsied andinfiltrated lymphocytes can be removed. A gene for a tumorcidal agent,such as TNF in a retroviral expression vector, is then used to transfectthe tumor infiltrating lymphocytes (TILs), which are then expanded withIL-2. When the transfected TILs are injected back into the patient, theTILs are specifically directed to the original tumor and migrate backinto the tumor, where the tumorcidal gene product is released for localeffect. (See, Thomas and Sikora, 1989.) Since all ELAMs bind some formof leukocyte and thereby mediate infiltration, modified TIL therapies inwhich infiltrated leukocyte cells are isolated, transfected forexpression of a particular desired gene product, amplified andreintroduced to the patient are contemplated herein.

An alternative TIL therapy takes advantage of the fact that certain celltypes, notably some forms of cancer cells, express ELAMs or MILAs. Forexample, colon carcinomas are known to express CDX and melanomas expressVLA4.

Employing the DNA sequences disclosed herein, a therapy can be designedto enhance and improve the cytolytic activity of leukocytes bytransfecting them to express surface ELAMs or MILAs, thereby improvingtheir binding to target cells expressing the corresponding ligand. Wherethe cytolytic activity of a leukocyte cell type is increased as afunction of stronger cell-cell adhesion, such a method would improve theability of leukocytes to destroy targeted cells. For example, in thecase of colon carcinoma or melanoma, leukocytes (preferably infiltratingleukocytes, which already have an affinity for the target cancer cell)may be transfected with an expression vector including a gene for ELAM1(in the case of colon carcinoma) or VCAM1 or VCAM1b (in the case ofmelanoma). Introducing such leukocytes into the patient provides apopulation of leukocytes capable of homing in on the carcinoma ormelanoma cells, respectively, which leukocytes have enhanced ability toadhere to those cells to produce the desired cytolytic effect.

We have also found that incubating HUVECs with TNF and IFN-γ togetherincreases VCAM1 expression about one-hundred percent over incubationwith TNF alone. Activated T cells secrete IFN-γ, and therefore maypromote their own recruitment to inflammatory sites through a positivefeedback system: VCAM causes T cell binding, T cells further stimulateVCAM production via IFN-γ secretion. Thus, we have devised a newtreatment for VCAM-dependent pathologies which involves inhibition ofthis feedback mechanism. The treatment comprises inhibiting cytokinessuch as IL-1, TNF or IFN-γ, for example with monoclonal antibodies, toblock cytokine-stimulated production of VCAM.

We have also isolated a MILA, CDX, that is involved in ELAM1-mediatedadhesion and, in fact, is probably the (or an) ELAM1 ligand. Theisolation involved, as a first step, the production of monoclonalantibodies against the CDX molecule. We immunized mice with whole HL-60cells, a PMN-related cell line, that was known to bind to ELAM1.Alternatively, one could immunize with any cell line that binds toELAM1, including PMNs themselves and, as we shall show, U937 cells. Inaddition, to isolate MILAs involved in adhesion to other ELAMS, onecould immunize with any cell line that binds to the appropriate ELAM.For example, in isolating VCAM1, we have identified two such cell lines:The B-lymphocyte-like cell line, RAMOS, and the T-lymphocyte-like cellline, JURKAT.

After finding that immune serum from the immunized mice inhibitedbinding of HL-60 cells to HUVECs in the adhesion assay we will describe,we created hybridomas from spleen cells in a manner well known to theart. (Goding, 1983.) Then we identified those hybridomas that producedmonoclonal antibodies against CDX by testing their ability in theadhesion assay to inhibit binding of HL-60 cells to induced HUVECs. Weused several of these hybridomas to produce ascites fluid containingmonoclonal antibodies.

One can also generate monoclonal Fab fragments recognizing theseantigens using the technique of Huse et al. (1989). (See also Skerra andPluckthun, 1988.) Alternatively, one can produce single domainantibodies as described by Ward et al. (1989).

Our monoclonal antibodies against CDX possess the followingcharacteristics: First, they inhibit binding of HL-60 cells or PMNs tocells that express ELAM1. Second, these antibodies exhibit a specificcell-binding pattern—they recognize cells that bind to ELAM1, but theydo not recognize cells that do not bind to ELAM1. Third, they have arecognition pattern for human cell lines that is distinct from thepattern of antibodies against other cell-surface molecules, such asanti-LFA-1, anti-LFA-3, anti-CD44, anti-ICAM, anti-CD4, and anti-Leu8.

We used these Moabs to isolate CDX. We radioactively labeled HL-60surface proteins and surface proteins from neutrophils (isolated fromhuman blood) with iodine using a modification of a method described byKurzinger (Kurzinger et al., 1981) or metabolically with ³⁵S-methionine.We solubilized the membrane proteins and incubated them with an anti-CDXmonoclonal bound through a μ-chain-specific rabbit anti-mouse IgG toProtein A sepharose (ARX), and then we isolated the antibody-boundprotein. This protein is CDX isolated substantially free of normallyassociated animal proteins. The protein appears on SDS-PAGE as a single,diffuse band of about 150 kD. A 90 kD protein band was sometimesobserved in the bound proteins from HL-60 cells and always in theproteins from neutrophils. We believe this 90 kD band represents a CDXdegradation product. We also sometimes observed higher molecular weightbands (i.e., around 170 kD). These may be non-specific bands. When theisolated 150 kD protein was treated with N-glycanase, the molecularweight was reduced to approximately 70 kD. When the 150 kD band wastreated with N-glycanase and O-glycanase, the molecular weight was notfurther reduced. We believe this represents the protein core of a veryheavily glycosylated protein.

We have isolated two DNA sequences, clone 7.2 and clone 1, that appearto encode 1,3-fucosyl transferases that glycosylate the CDX polypeptideand impart to it the ability to bind ELAM1. 1,3-fucosyl transferases arehighly specific enzymes that function in the Golgi apparatus andendoplasmic reticulum to attach fucosyl moieties to appropriate acceptorcarbohydrates through a 1,3 glycosidic linkage. The genetic structure ofthese sequences is consistent with that of other, known glycosyltransferases. Furthermore, CHO cells transfected with clone 7.2 expressfucosyl transferase activity.

Several 1,3-fucosyl transferases are known to the art. (Paulson andColley, 1989 and Kukowska-Latallo et al., 1990.) These proteins ofsimilar activity share little sequence homology between themselves orother glycosyl transferases. (Paulson and Colley, 1989 andKukowska-Latallo et al., 1990.) Therefore, we would not expect these DNAsequences to share homology with the DNA sequences of this invention.However, other species are likely to contain homologous genes that sharesignificant sequence homology with the DNA sequences disclosed here. Onecan isolate these homologous genes using the DNA sequences of thisinvention as probes under standard hybridization conditions. Thisinvention specifically contemplates and encompasses such sequences.

When COS 7 cells were transfected with either of these two clones, theybehaved like cells expressing CDX, that is, they became “visible” toELAM1 in that they were able to produce a surface glycoprotein to whichELAM1 binds and which are recognized by the α-CDX monoclonal, SGB₃B₄.Using α-CDX monoclonals, we immunoprecipitated a 130 kD glycoproteinfrom transfected COS cells, which we have designated Pseudo-X.Similarly, CHO cells transfected with clone 7.2 also became visible toELAM1 and α-CDX. They express a 140 kD glycoprotein which we havedesignated Pseudo-X₂.

Neither Pseudo-X nor Pseudo-X₂ are CDX. Pseudo-X has a molecular weightof about 130 kD and Pseudo-X₂ of 140 kD. CDX has a molecular weight of150 kD. When treated with N-glycanase or hydrofluoric acid (whichremoves all carbohydrate), Pseudo-X shifts to 110 kD. Pseudo-X₂ shiftsto approximately 120 kD. CDX shifts to about 70 kD. Neither migrates at46 kD or 59 kD, the predicted molecular weights of protein 7.2 andprotein 1. Pseudo-X and CDX also have different V8 and chymotrypsindigestion patterns.

We isolated clone 7.2 and clone 1 as follows: We created a cDNA libraryfrom mRNA of a human cell line, HL-60, that expresses CDX. We enrichedthis library by using subtraction techniques, as we describe below, witha human cell line that does not express CDX, in this case HeLa cells.This produced a subtracted library containing about 2100 clones. Wetransfected a monkey kidney cell line, COS 7, with the subtractedlibrary which we assayed in a number of ways.

We incubated the transfected cells with the α-CDX monoclonal antibodiesand panned them on plates coated with anti-mouse IgG or IgM (Wysocki andSato, 1978); cells binding to the plates would be those expressing amolecule recognized by α-CDX Moabs. In this manner, we identifiedadherent cells transfected with a 2.1 kb DNA insert. We subcloned aportion of this sequence into a sequencing vector and designated itpSQ219. The DNA insert in the pCDM8 clone was designated clone 7.2. Wealso isolated a 2.9 kb insert by hybridization, which we designatedclone 1. These two clones encode protein 7.2 and protein 1,respectively.

We are also isolating a DNA sequence that codes on expression for CDXusing techniques known to the art. Some practical techniques involveusing expression systems to express cloned DNA. As we have mentioned, avariety of eukaryotic expression systems are available.

One can isolate a DNA sequence encoding CDX using antibodies thatrecognize the CDX polypeptide, rather than the CDX glycoprotein. Theseantibodies are used to probe an HL-60 cDNA library like the one wedescribed above.

Another method for isolating a DNA sequence encoding CDX (or anotherMILA) would employ fluorescent-antibody labeling. In this method,CDX-expressing cells are incubated with α-CDX Moabs and then the Moabsare labeled with, e.g., fluorescently tagged anti-mouse antibody. Cellsbinding the fluorescent antibodies may then be sorted with afluorescence activated cell sorter (FACS). The DNA from the sorted cellsmay be used to transform a bacterial host such as E. coli. DNA from theresulting colonies may then be used to transfect COS 7 cells, and thisprocedure may be repeated until a single CDX-expressing clone isidentified.

A third method is to pan the transfected cells as described above onplates coated with recombinant soluble ELAM1 (rsELAM1). We describe amethod to coat plates with rsELAM1 in Example VIII. Cells binding to theplates will be those expressing CDX. Other soluble ELAMs can similarlybe used to isolate cells expressing their ligands or MILAs involved intheir adhesion pathways.

An expression library may also be created in E. coli. For example, a λZAP® (Stratagene)/HL-60 library may be constructed and used to expressthe inserted DNA in E. coli. After plating, the plaques can be directlyscreened with, e.g., radioactively labeled α-CDX monoclonals. (Young andDavis, 1983 and Young and Davis, 1984.) The plaques to which themonoclonals bind can be picked and the DNA insert isolated from them.

Another method we are using to identify ELAM ligands, not based onantibody recognition, is to transfect COS 7 cells with an appropriatelibrary, that may be subtracted, and then pan them directly ontoELAM-expressing cells (such as induced HUVECS, ELAM-expressing COS 7cells, or ELAM-expressing CHO cells). Once again, multiple rounds ofpanning are required to enrich the library sufficiently to isolate thepertinent clones.

Another technique for isolating the DNA sequences coding for CDX (orother MILAS) involves screening a cDNA library with oligonucleotideprobes. By purifying a sufficient quantity of CDX, for example byaffinity chromatography using immobilized antibody to CDX or immobilizedELAM1, one may determine a partial amino acid sequence and synthesizeoligonucleotide probes that correspond to at least a portion of the CDXgene. These probes may then be used to screen the cDNA library.Alternatively, the oligonucleotides may be used as primers to generatelong probes to be used in screening the library for CDX (MILA) genes.

We have also identified a ligand for VCAM1 and VCAM1b. It is theintegrin VLA4. (Hemler, 1988; Hemler et al., 1987a; and Hemler et al.,1987b.) The integrins are a group of cell-extracellular matrix andcell-cell adhesion receptors exhibiting an αβ heterodimeric structure.(Hynes, 1987; Marcantonio and Hynes, 1988.) Investigators haveidentified three subfamilies of integrins categorized according to the 8subunit. The VLA (Very Late Antigen) proteins belong to the β₁subfamily, many of whose members are specialized for cell-extracellularmatrix attachment. (Hynes, 1987 and Ruoslahti, 1988.) VLA4 is expressedin relatively high levels on lymphoid cells (such as B and T cells) andmyeloid cells, but is hardly detectable in other cells (Hemler et al.,supra.) The binding of B and T cells to the extracellular matrix ismediated by VLA4 and its ligand, human fibronectin (FN). (Wayner et al.,198′.) The discovery that VLA4 is a ligand for VCAM1 is importantbecause it now defines one binding pathway of B and T lymphocytes toactivated endothelial cells. Therefore, we describe the use of VLA4 andVCAM1 and 1b as ligand and receptor in the methods described below.

We contemplate several uses for ELAM and MILA DNA sequences andmolecules in the present invention. First, one may use ELAMs and MILAsto produce monoclonal antibody preparations that are reactive for thesemolecules. The Moabs may be used in turn as therapeutic agents toinhibit leukocyte binding to endothelial cells.

Second, one may use a soluble form of ELAM, soluble ELAM ligand, orfragments of either as binding inhibitors. The ELAM peptides would bindto the ELAM ligand on leukocytes, and the ELAM ligand would bind to ELAMon endothelial cells. Both methods would thereby inhibit leukocytebinding to endothelial cells. To produce recombinant soluble ELAM(rsELAM) or rsELAM ligand one preferably would alter a DNA encodingthose molecules to eliminate the transmembrane region. Thus, DNAs forsoluble molecules would include all or part of the extracellular domain,perhaps attached to the cytoplasmic domain. This approach has alreadybeen validated using soluble CD4, the surface protein on T-cells thatbinds to the AIDS virus. (Fisher et al., 1988.) This approach alsoavoids the problems of antibody therapy, since the polypeptides usedwould be less likely to induce an immune response.

One problem investigators have encountered with soluble recombinantmolecules is a short in vivo plasma half-life. (Capon et al., 1989.)Because such molecules are quickly cleared from the system, large dosesor frequent injections are necessary to have a therapeutic effect.Therefore, investigators have sought methods to increase the half-lifeof soluble molecules. A potential solution is to link the solublemolecule to another molecule known to have a longer half-life in theblood stream. Due to their long half life, immunoglobulin molecules arepromising candidates. Capon et al. (1989) have described the linking ofsoluble CD4 to an immunoglobulin molecule using recombinant DNAtechniques. In this approach, one replaces the variable region of animmunoglobulin molecule with the soluble protein, forming aprotein/immunoglobulin fusion protein.

It is expected that the rsELAM/immunoglobulin fusion proteins will havegreater plasma half-life than rsELAM alone. Such fusion proteins arepreferably produced with recombinant constructs, fusing a DNA sequenceencoding the soluble molecule to a DNA sequence encoding the constantdomain of an immunoglobulin molecule. The recombinant DNA may then beexpressed in an appropriate host cell, preferably an animal cell, toproduce the fusion protein.

We expect ELAM/immunoglobulin fusion proteins to have another advantage.Because immunoglobulin molecules are normally bivalent (i.e., they havetwo binding sites) an ELAM/immunoglobulin fusion protein would have twoELAMs and so, two ELAM ligand binding sites. Therefore, one would expectthem to have greater affinity or avidity for cells displaying ELAMligands.

Third, one may use molecules binding to ELAMs (such as anti-ELAMantibodies, or markers such as the ligand or fragments of it) to detectinflammation. This involves, for example, making a molecule detectableby fluorescence or radioactivity, administering it to a patient anddetermining where in the body it accumulates. In this way one could alsoidentify the type of inflammation. For example, binding to ELAM1 wouldindicate acute, as opposed to chronic inflammation.

Fourth, if an ELAM binds to its ligand through a carbohydrate moiety orsome other post-translational modification, one could use ELAM toidentify the carbohydrate on the ELAM ligand to which it bound.

Fifth, one could use ELAMs and MILAs as part of a system to screen smallmolecules for adhesion inhibitors. For example, one could create anassay system in which small molecules are tested for the ability toinhibit the interaction between CDX and ELAM1. Small molecule inhibitorsidentified in this way would provide candidates for anti-inflammatorydrugs.

Sixth, one could use these molecules to identify endogenous proteinsthat inhibit leukocyte binding to ELAMs. Investigators have tentativelyidentified one such molecule, leukocyte adhesion inhibitor (LAI), thatis involved in detaching bound PMNs from endothelium. (Wheeler et al.,1988.)

Seventh, one can generate VCAM/ICAM fusion proteins. We know that bothproteins are composed of several structural domains. (Simmons et al.,1988.) DNA sequences encoding various domains of each protein are fusedusing, for example, the genetic fusion techniques we describe for makingELAM/immunoglobulin fusion proteins. The domains chosen are those havingthe ability to bind VCAM1 or VCAM1b ligands and ICAM1 ligands,respectively. Domains binding VLA4 and LFA1, the known ligands, arepreferable. The polypeptides produced on expression of these DNAsequences are useful because they would block adhesion of any cellhaving a ligand to either VCAM1 or VCAM1b, or ICAM1 or both.

Finally, one could use ELAM and ELAM ligand DNA sequences to producenucleic acid molecules that intervene in ELAM or ELAM ligand expressionat the translational level. This approach utilizes antisense nucleicacid and ribozymes to block translation of a specific mRNA, either bymasking that mRNA with an antisense nucleic acid or cleaving it with aribozyme. These methods will be useful in treating inflammatoryconditions.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into ELAM-producing cells. Antisense methods have been used toinhibit the expression of many genes in vitro. (Marcus-Sekura, 1988;Hambor et al., 1988.)

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it. (Cech, 1988.)Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, 1988.) Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen-base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave, mRNAs for ELAM1, VCAM1 andVCAM1b, CDX and VLA4.

Antisense molecules and ribozymes may be used in methods to treatinflammation by introducing into cells molecules that interfere with theexpression of adhesion molecules. Since ELAMs are induced on endothelialcells during inflammatory episodes, and since therapeutic agents can bedelivered to vascular endothelium easily by intravenous injection,endothelial cells are attractive targets for such therapies, providedthe antisense molecules or ribozymes can be delivered effectively to theappropriate cells.

Investigators have suggested two approaches which could be used todeliver these molecules to target cells. The first involves transfectingthe target cell with a vector that expresses the anti-ELAM antisensenucleic acid or the ELAM-specific ribozyme as an mRNA molecule. (Hamboret al., supra.) While this approach is very useful when dealing withcell lines in vitro, it may not be as effective in vivo. A secondapproach that is more promising for in vivo delivery involves loadingliposomes with anti-ELAM antisense molecules, ELAM-specific ribozymes orvectors which express them. These liposomes could also contain anti-ELAMmonoclonal antibodies to direct the liposome to sites of inflammation.This form of delivery would provide a negative feedback system, sinceappearance of an ELAM on a cell would make the cell a target forsuppression; and successful penetration of the antisense or ribozymecomponent would halt ELAM production, thereby eliminating the cell as atarget.

Another feature of this invention is the expression of the ELAM, MILAand other DNA sequences disclosed herein. As is well known in the art,DNA sequences may be expressed by operatively linking them to anexpression control sequence in an appropriate expression vector andemploying that expression vector to transform an appropriate unicellularhost.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, PCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAs, e.g., the numerous derivatives of phage A, e.g., NM989, andother phage DNA, e.g., M13 and Filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyand late promoters of SV40 or adenovirus, the lac system, the trpsystem, the TAC or TRC system, the major operator and promoter regionsof phage λ, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase (e.g., PhoS), the promoters of the yeast α-matingfactors, and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B—W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

It will also be recognized that expression of the DNA sequences of thepresent invention may have different effects in different hosts. Forexample, whereas clone 7.2 expressed in COS cells leads to theappearance of an ELAM1-binding surface molecule, expression of clone 7.2in, e.g., prokaryotic host cells may have no similar effect, sinceprokaryotes lack internal cell structures (e.g., Golgi apparatus) thatmay be necessary for the biological functionality of protein 7.2. On theother hand, for isolation and purification of the clone 7.2 expressionproduct intact, host cells in which protein 7.2 does not have a functionin the cellular biochemistry (such as the catalytic role of a glycosyltransferase) may be preferred. The practitioner will be able to selectthe appropriate host cells and expression mechanisms for a particularpurpose.

Considering these and other factors, a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

Several strategies are available for the isolation and purification ofprotein 7.2 and protein 1 after expression in a host system. One methodinvolves expressing the proteins in bacterial cells, lysing the cells,and purifying the protein by conventional means. Alternatively, one canengineer the DNA sequences for secretion from cells. For example, Colleyet al. (1989) describe purifying a sialyltransferase by engineering thecleavable signal peptide of human gamma-interferon onto the DNA sequencefor the transferase. Larsen et al. (1990) fused the DNA sequence forprotein A to the amino-terminal end of a fucosyl transferase gene andexpressed it as an excreted fusion protein. In these constructions, onecan optionally remove the transmembrane region of these proteins thatexist near the amino terminus. After secretion the proteins are purifiedfrom the medium. Similar strategies are available for bacteria.

Increasingly scientists are recognizing the value of enzymes ascatalysts in organic synthesis. (Wong, 1989.) The 1,3-fucosyltransferases of this invention are useful for enzymatic synthesis ofcarbohydrates in vitro. Specifically, they are useful for catalyzing thelinkage of fucose to appropriate acceptors through a 1,3 glycosidicbond. We describe one set of suitable conditions for this catalysis inExample XII, relating to an assay for fucosyl transferase activity. Oneskilled in the art will recognize other suitable conditions under whichthe 1,3 fucosyl transferases described herein may be advantageouslyemployed.

It is now clear that the carbohydrate moiety of CDX is important inELAM1-mediated cell adhesion. A molecule comprising the carbohydratemoiety of CDX, Pseudo-X or Pseudo-X₂, or a fucose-containing portion ofthat moiety may be sufficient to function as an ELAM1 ligand. Suchmolecules may be useful in methods, including therapies, directed toinhibiting ELAM1-mediated cell adhesion.

This invention is also directed to small molecules that inhibit theactivity of the 1,3-fucosyl transferases described herein, includingsynthetic organic chemicals, natural fermentation products, peptides,etc. These molecules may be useful in therapies aimed at inhibitingELAM1-mediated cell adhesion. To identify such molecules, one produces atest mixture by contacting together an inhibitor candidate, a fucoseacceptor and a 1,3-fucosyl transferase. The fucose acceptor is,preferably, LacNAc or 2′-fucosyllactose. The 1,3-fucosyl transferasepreferably is derived from an extract from a cell transformed with clone7.2 or clone 1. Then one assays the test mixture for 1,3-fucosyltransferase activity, such as described in Example XII.

The existence of antibodies against ELAM1, VCAM1 and 1b, CDX and VLA4makes possible another method for isolating other ELAMs and ELAMligands. The method takes advantage of an antibody characteristic knownas idiotypy. Each antibody contains a unique region that is specific foran antigen. This region is called the idiotype. Antibodies, themselves,contain antigenic determinants; the idiotype of an antibody is anantigenic determinant unique to that molecule. By immunizing an organismwith antibodies, one can raise “anti-antibodies” that recognize them,including antibodies that recognize the idiotype. Antibodies thatrecognize the idiotype of another antibody are called anti-idiotypicantibodies. Some anti-idiotypic antibodies mimic the shape of theoriginal antigen that the antibody recognizes and are said to bear the“internal image” of the antigen. (Kennedy, 1986.) When the antigen is aligand, certain anti-idiotypes can bind to that ligand's receptor.Investigators have identified several of these, including anti-idiotypesthat bind to receptors for insulin, angiotensin II, adenosine I,β-adrenalin, and rat brain nicotine and opiate receptors. (Carlsson andGlad, 1989.)

Taking advantage of this phenomenon, other ELAMs and ELAM ligands may beisolated using anti-idiotypic antibodies. Anti-idiotypes may be used toscreen for molecules binding to the original antigen. For example, onemay use this technique to identify other ELAM ligands.

We have demonstrated that related ELAMs exist with similar domainstructures (i.e., VCAM1 and VCAM1b.) As a result of gene shuffling,there may be several adhesion molecules on the cell surface that shareone or more domains. Anti-idiotypic antibodies, which recognize anyshared domains, are useful to isolate immunochemically ELAMs orELAM-ligands not identified by bioassay, which relies on the protein'sfunction, rather than structure.

In order that one may better understand this invention, we set forth thefollowing examples. These examples are for purposes of illustration andare not to be construed as limiting the scope of the invention in anymanner.

EXAMPLE I Preparation of a cDNA Sublibrary Enriched for ELAM Sequences

We prepared a cDNA sublibrary enriched for ELAM sequences as follows:

We isolated human umbilical vein endothelial cells (HUVECs) fromumbilical cords, grew the cells in primary culture, and seriallypassaged them as described in Gimbrone (1976). We used HUVECs forlibrary construction at passages 4 or 5. To induce the cells to producemRNA for ELAMs we incubated confluent monolayers for 2.5 hours at 37° C.with recombinant human IL-1β (10 units/ml). We isolated the mRNA fromthese cells and reverse-transcribed it into cDNA using techniques wellknown to the art. (Gubler and Hoffman, 1983.) Using standard procedures,we ligated double stranded cDNA to a NotI-BstXI linker/adaptor havingthe following sequence: 5′ GCG GCC GCT TTA GAG CAC A 3′ 3′ CGC CGG CGAAAT CTC       5′We then size-selected the cDNA on a 4.2 ml 5-20% potassium acetategradient, 2 mM EDTA, 1 μg/ml ethidium bromide, in a Beckman® SW60 Rotorfor 3 hours at 50,000 rpm at 22° C. according to the protocols of BrianSeed. (See also Maniatis, 1982, p. 278.) We pooled the cDNA fragments ofgreater than 500 base pairs. Then we prepared the vector, pCDM8 (a giftfrom Brian Seed). We digested this plasmid with BstXI. To remove the 400base pair stuffer fragment we centrifuged the mixture on a potassiumacetate gradient, as above, and isolated the large fragment. We furtherpurified this fragment by agarose gel electrophoresis, and then ligatedthe cDNA to the vector. In this way we created recombinant DNA moleculescontaining DNA sequences for mRNA expressed in induced HUVECs. We usedthese plasmids to transform E. coli MC1061 P3. The result was acollection of over 7×10⁶ recombinant clones comprising a cDNA libraryfor IL-1β-induced HUVEC mRNA.

In order to prepare from this cDNA library a sublibrary enriched forELAM cDNA sequences, we first prepared a subtracted probe enriched forELAM sequences. We prepared cDNA as above from HUVECs induced with IL-1βand labeled it with ³²P. (Davis, 1986.) Then we isolated mRNA fromHUVECs that had not been induced. To subtract uninduced cDNA sequencesfrom induced sequences we hybridized the mRNA with the cDNA and isolatedcDNA that had not hybridized to mRNA, as described by Davis (1986). Wesubjected the isolated cDNA to another round of subtraction to increasethe level of enrichment. In all, we prepared three batches of subtractedprobes enriched for ELAM sequences.

We tested the level of purification of the probe by Northern blot.(Lehrach et al., 1977.) We ran a gel with parallel lanes of polyA+ mRNAfrom induced and uninduced HUVECs and blotted it on Gene Screen® (NewEngland Nuclear). Hybridization and subsequent autoradiography revealedthat the probe bound strongly to a 4 kb band in the induced lane but didnot bind, beyond background, to the uninduced lane. Occasionally wenoted less intense hybridization bands to other messages in the inducedlane.

We used the subtracted probe to create a cDNA sublibrary in E. coliMC1061 P3 enriched for IL-1β induced sequences. We began by plating-outone million clones of the IL-1β-induced HUVEC cDNA library. We platedone million colonies on Gene Screen Plus® filters (New England Nuclear)on LB agar containing 12.5 μg/ml ampicillin and 7.5 μg/ml tetracycline,and grew them at 37° C. for 12 hours. We made two replicate filters(lifts) from each master. We grew these on LB agar containing 12.5 μg/mlampicillin and 7.5 μg/ml tetracycline for 4 hours and amplified them onLB agar containing 250 μg/ml chloramphenicol for 16 hours. We lysed thefilters according to manufacturer's protocol and then prehybridized themin Plaque Screen® Buffer (0.05M TRIS-HCl pH7.5, 1N NaCl, 1% SDS, 0.1%sodium pyrophosphate, 0.2% polyvinylpyrrolidone (PVP), 0.2% Ficoll-400,0.2% BSA). We hybridized the filters at 65° C. for 40 hours in 50 mlPlaque Screens Buffer containing 10% dextran sulfate and 100 μg/ml yeasttRNA and approximately 1×10⁷ cpm of the subtracted IL-1β-induced HUVECcDNA. We then washed the filters twice with Plaque Screen® Buffer, twicewith 2×SSC, 1% SDS, and twice with 1×SSC, 1% SDS at 65° C. We thenexposed the filters to film for 5 days.

We selected colonies that hybridized to the probe by aligning the masterfilters with the autoradiographs and scraping the colonies off thefilters with sterile toothpicks. We placed each scraping in one well ofa 96-well microtiter plate filled with LB broth containing 7.5 μg/mltetracycline and 12.5 μg/ml ampicillin. After inoculation, we incubatedthe microtiter plates overnight at 37° C. When the cells had grown weadded glycerol to each well to a final concentration of 20% and storedthe plates at −70° C. In this way we isolated from the master libraryfilters 864 colonies comprising the cDNA sublibrary enriched for ELAMsequences. We point out that because of the plating density, not all thecolonies of the enriched sublibrary were pure.

We carried out two sets of procedures in parallel with the enriched cDNAsublibrary.

EXAMPLE II Isolation of a Clone Expressing ELAM1

In a first procedure we isolated from the enriched sublibrary a cloneexpressing ELAM1. We chose to transfect this sublibrary into a cell linecompetent for high-level transient expression, the African Green Monkeykidney cell line, COS 7. We plated the cells and transfected thesublibrary by spheroplast fusion. (Sandri-Goldin et al., 1981.)Forty-eight hours after transfection, we assayed the COS 7 cells forexpression of ELAM1 by their ability to bind HL-60 cells, a cell lineknown to bind to endothelial cells stimulated with inflammatory agents.

We performed the assay as follows: We labeled HL-60 cells withcarboxyfluorescein diacetate according to the Brenan and Parish method.(Brenan and Parish, 1984.) Briefly, we resuspended HL-60 cells inRPMI/10% FCS at a concentration of 1×10⁷ cells/ml, and addedcarboxyfluorescein diacetate to a final concentration of 0.1 mg/ml froma stock solution of 10 mg/ml in acetone. We incubated COS 7 cells withlabeled HL-60 cells for 15 minutes at room temperature. We washed thecells 3-4 times with RPMI/1% FCS. We examined the petri dish byfluorescence microscopy for clusters of adherent HL-60 cells. We pickedregions of the cell plates with clusters of HL-60 cells and lysed thecells in 0.6% SDS, 10 mM EDTA, pH 8, then rescued the plasmids accordingto the method of Hirt. (Hirt, 1967.) We used these pooled plasmids totransform E. coli MC1061 P3. We grew colonies from these transformantsand performed a second round of spheroplast fusion with COS 7 cells withsubsequent assay for HL-60 adhesion. From among the cells that werepositive for adhesion we selected one and isolated the plasmid from it.We designated a culture containing this plasmid ELAM pCDM8 clone 6. Wedeposited this plasmid under the Budapest Treaty with In VitroInternational, Inc., 611 P. Hammonds Ferry Rd., Linthicum, Md., 21090(USA) on Apr. 20, 1989. It is identified as:

-   -   ELAM pCDM8 clone 6/E. coli MC1061 P3    -   Accession Number IVI-10204

EXAMPLE III Isolation of cDNA Inserts for ELAM1 Sequences

In a second procedure, we isolated cDNA inserts for IL-1β-induced cDNAsequences. We selected at random twenty-four of the 864 colonies of theenriched library and isolated plasmids from them using the alkalineminiprep procedure of Maniatis.

(Maniatis, 1982.) We digested the plasmid DNA with XhoI or NotI andseparated the fragments on 1% agarose gels. We identified from this geltwo plasmids with inserts of greater than 3 kb, isolated these insertsand labeled them with 2P. (See, Feinberg and Vogelstein, 1983 and 1984.)

We then performed Northern blots with these inserts, as described above.Both inserts hybridized to bands at 4 kb in the induced HUVEC mRNA lanebut did not hybridize to the uninduced HUVEC mRNA lane. The insertscross-hybridized with the ELAM1 expressing plasmid ELAM pCDM8 clone 6(described above) as well. We subcloned these inserts into NotI-digestedpNN11 that had been treated with calf intestinal alkaline phosphatase.We constructed the sequencing plasmid pNN11 by removing the syntheticpolylinker from the commercially available plasmid PUC8 (Pharmacia PLBiochemicals) by restriction digestion and replacing it with a newsynthetic segment. The 2.5 kb backbone common to the pUC plasmids, thatprovides an origin of replication and confers ampicillin resistance,remained unchanged. The novel synthetic portion of pNN11 is shown inFIG. 2. We called these new constructs pSQ148 and pSQ149, respectively.

EXAMPLE IV A DNA Sequence for ELAM1

We determined the entire DNA sequence for the inserts of plasmids pSQ148and pSQ149 and 624 nucleotides of the sequence at the 5′ end of theinsert of ELAM pCDM8 clone 6. We used the Maxam-Gilbert method. (Maxamand Gilbert, 1980.) Because the sequences have significant overlap, weobtained a composite sequence of ELAM cDNA, a sequence of 3863nucleotides. This sequence consists of 140 nucleotides of the 5′untranslated region, 1830 nucleotides encoding 610 amino acids, and 1893nucleotides of the 3′ untranslated region (including a translationalstop codon and a polyadenylation signal). The mature protein derivedfrom the deduced amino acid sequence has been designated ELAM1, and thecoding sequence has been designated the ELAM1 DNA sequence. The cDNAsequence of ELAM1 is shown in FIG. 1.

A search of the Genbank Data Base, release 58, December 1988, revealedthat the DNA sequence for ELAM1 has no significant homologies to knownDNA sequences.

We used this cDNA sequence to deduce the ELAM1 amino acid sequence, thatis also presented in FIG. 1. Our analysis of the sequence revealed thefollowing properties: The protein possesses a hydrophobic N-terminalsequence characteristic of a signal sequence. (von Heijne, 1986.) Wehave not yet determined the signal cleavage site and the matureN-terminus through protein sequencing, however based on von Heijne wepredict that the mature N-terminal amino acid will be tryptophan, atnucleotide number 204 in FIG. 1. The extracellular domain of thepolypeptide is approximately 554 amino acids including the signalsequence and is followed by a hydrophobic transmembrane region of 24amino acids. The protein possesses a short, charged cytoplasmic tail of32 amino acids. We note that the protein is cysteine-rich and containseleven potential N-glycosylation sites.

When we compared the amino acid sequence of ELAM1 to other proteins inthe NBRF and NEW protein data bases we found significant homology withseveral proteins, including complement C2 precursor, β-2-glycoprotein I,C4b-binding protein, complement factor B, complement factor H,Drosophila notch protein, the IgE receptor Hepatic lectin, andCoagulation factors IX and X precursors. Thus, we can divide ELAM1 intoat least three domains based on homology to the above-mentionedproteins: (1) a lectin-like domain (nucleotides 204-563 of FIG. 1); (2)an EGF-like domain (nucleotides 564-668); and (3) a consensus cysteinerepeat unit of 59-63 amino acids containing six cysteine residues perrepeat (nucleotides 669-1793). Other invariable amino acids in eachrepeat are proline, glycine, and tryptophan.

EXAMPLE V Monoclonal Antibodies Recognizing ELAM1

To make monoclonal antibodies that recognize ELAM1 we preparedhybridomas in essentially the same manner as we did in Example X, infra.However, we immunized the mice with ELAM1-expressing COS cells andidentified mice producing anti-ELAM1 antibodies by testing theirantiserum for the ability to block HL-60 cell adhesion to IL-1β inducedHUVECs.

We screened hybridomas produced in this manner for those producinganti-ELAM1 monoclonals using several assays. First, we tested theculture supernatants for antibodies having the ability to bind to a cellline that stably expressed ELAM1. This cell line was a line of CHO-DHFR⁻cells transfected with the ELAM1 animal cell expression vector,pBG341jod.ELAM. We created this plasmid by introducing the DNA sequenceencoding ELAM1 from pCDM8 clone 6 into the NotI site of pBG341.jod(described in Example VIII, infra). The ELAM1 expressing CHO-DHFR⁻derived cell line was detected using an adhesion assay to HL-60 cells.

Second, we screened hybridoma culture supernatants for the ability tobind cytokine-induced, but not control, HUVECs.

Third, we tested them for their ability to inhibit HL-60 cell adhesionto cytokine-induced HUVEC monolayers.

We identified one hybridoma clone, BB11, which produced a positiveresult in all three assays. BB11 immunoprecipitates proteins withmolecular weights of about 110 kD and 96 kD from ELAM1-expressing HUVECsand COS cells, representing variably glycosylated forms of ELAM1.(Bevilacqua et al., 1989.) It also completely blocked adhesion of HL-60cells to ELAM1-expressing COS and CHO cells. It produced immunoglobulinsof the IgG₂b class. We deposited a subclone of this hybridoma under theBudapest Treaty with In Vitro International, Inc., 611 P. Hammonds FerryRd., Linthicum, Md. 21090 (USA) on Dec. 13, 1989. It is identified as:

-   -   Monoclonal antibody CDB.BB11.BC6    -   Accession Number IVI-10220.

EXAMPLE VI Isolation of Clones Expressing VCAM1 and VCAM1b

We have also characterized and cloned two different ELAMs that bind tolymphocytes and lymphocyte-like cell lines. As a first step, wecharacterized the binding pathways of RAMOS, a B-lymphocyte-like line,and JURKAT, a T-lymphocyte-like line, to HUVECs induced with IL-1β orTNF for 4, 24, or 48 hours. We found that both RAMOS and JURKAT bindingwas maximal at 4 hours after induction with either IL-1β or TNF, andbinding was maintained at 24 hours and 48 hours after induction. RAMOSbinding was temperature-sensitive, occurring at room temperature but notat 4° C. JURKAT binding was reduced but not completely eliminated at 4°C., and thus JURKAT exhibited both a temperature-sensitive andtemperature-insensitive component. Antisera from mice immunized withJURKAT cells inhibited binding from both JURKAT and RAMOS cells toHUVECs, indicating that RAMOS and JURKAT share a MILA. Neither RAMOS norJURKAT bound to COS or CHO cells expressing ELAM1, indicating thepresence of at least one other inducible ELAM on HUVECs, at 4 to 48hours after induction.

In order to isolate clones expressing the ELAMs involved in RAMOS andJURKAT binding to HUVECs, we screened the previously describedELAM-enriched HUVEC cDNA sublibrary by the method described in ExampleII, supra. We incubated carboxy-fluorescein diacetate-labeled RAMOS andJURKAT cells with sublibrary-transfected COS 7 cells. Regions of thecell plates with clusters of bound cells were picked and lysed, and theplasmids were rescued, transformed into E. coli, and reassayed in COS 7cells as previously described. Plasmids were isolated from individualbacteria colonies from the transformants that were positive on reassay.These plasmids were transfected individually into COS 7 cells, and aplasmid that tested positive for adhesion to RAMOS and JURKAT wasidentified. The cDNA insert from this plasmid was excised, radioactivelylabeled, and used to probe a Northern blot according to the proceduresof Lehrach (1979). The probe hybridized to an RNA species approximately3.4 kb in length. The RNA was undetectable in uninduced HUVEC RNA,barely detectable at 5, 10, 30 or 60 minutes after treatment with IL-1β,but abundant at 2, 24, 48 and 72 hours after treatment with IL-1β.

We designated the plasmid AM pCDM8 clone 41. We deposited this plasmidunder the Budapest Treaty with In Vitro International, Inc., Linthicum,Md. (USA) on May 24, 1989. It is identified as:

-   -   AM pCDM 8 clone 41/E. coli MC1061 P3    -   Accession Number IVI-10206

We have also isolated a cDNA for another VCAM. We screened theIL-1β-induced HUVEC cDNA library (Example I) with a labeledVCAM1-encoding insert from AM pCDM 8 clone 41. We sequenced one ofthese, clone 1E11. We found several clones that were longer than theclone 41 insert as analyzed by restriction mapping with XbaI. Wesequenced one of these, clone 1E11. We deposited it under the BudapestTreaty with In Vitro International, Inc., Linthicum, Md. (USA) on Dec.7, 1989. It is identified as:

-   -   VCAM 1B Clone 1E11 pCDM8/E. coli MC1061p3    -   Accession Number IVI-10216.

We are also isolating DNA sequences for other ELAMs. We are collectingmRNA from HUVECs around forty-eight hours after IL-1β induction. We willisolate the ELAM cDNA sequences in a manner similar to the one we usedto isolate the cDNA sequences for ELAM1 and VCAM1 and 1b.

Alternatively, one may identify other ELAMs by inducing cells with otherinflammatory agents, such as TNF, LT, LPS, interferons, or combinationsof such agents.

EXAMPLE VII DNA Sequences for VCAM1 and VCAM1b

We determined the entire DNA sequence for the insert of plasmid AM pCDM8clone 41 by the method of Maxam and Gilbert (1980). This sequenceconsists of 106 nucleotides of the 5′ untranslated region, 1941nucleotides encoding 647 amino acids, and 764 nucleotides of the 3′untranslated region including a translational stop codon. The proteinderived from the cDNA sequence has been designated VCAM1, and the codingsequence has been designated the VCAM1 DNA sequence. We have presentedthe cDNA sequence of VCAM1 in FIG. 3. The putative amino acid sequenceof VCAM1 is also indicated in FIG. 3.

We also determined the entire DNA sequence for the insert of plasmidVCAM1b pCDM8 1E11 by the method of Maxam and Gilbert (1980). Thissequence consists of 99 nucleotides of the 5′ untranslated region, 2217nucleotides encoding 739 amino acids and 764 nucleotides of the 3′untranslated region including a translational stop codon. We havedesignated the mature protein derived from the cDNA sequence as VCAM1band the coding sequence as the VCAM1b DNA sequence. We have presentedthe cDNA sequence and putative amino acid sequence of VCAM1b in FIG. 4.

Comparison of the DNA and amino acid sequences of VCAM1 and VCAM1brevealed that they are virtually identical except for one significantdifference: VCAM1b contains an insertion of 276 nucleotides near themiddle of the coding region. These nucleotides encode 92 additionalamino acids which form an extra domain of 84 amino acids situatedbetween the end of VCAM1 domain 3 and the beginning of VCAM1 domain 4.We discuss the significance of this domain, designated VCAM1 domain 3B,below.

Our analysis of the sequences revealed the following properties: TheVCAM1 polypeptide possesses a hydrophobic N-terminal sequencecharacteristic of a signal sequence. (von Heijne, 1986.) We have not yetdetermined the signal cleavage site and the mature N-terminus throughprotein sequencing, however based on von Heijne we predict that theN-terminal amino acid of the mature protein will be phenylalanine, atnucleotide number 179 in FIG. 3. The extracellular domain of thepolypeptide is approximately 606 amino acids including the signalsequence and is followed by a hydrophobic transmembrane region of 22amino acids. The protein possesses a short, charged cytoplasmic tail of19 amino acids. We note that the protein contains six potentialN-glycosylation sites.

Similarly, the N-terminal amino acid of the mature VCAM1b protein shouldbe the phenylalanine, at nucleotide number 172 of FIG. 4. Theextracellular domain of the polypeptide, which is longer than VCAM1, isapproximately 698 amino acids including the signal sequence and isfollowed by a hydrophobic transmembrane region of 22 amino acids. Theprotein possesses a short, charged cytoplasmic tail of 19 amino acids.We note that the protein contains seven potential N-glycosylation sites.

Comparison of the amino acid sequences of VCAM1 and VCAM1b with otherproteins in the NBRF and NEW protein databases revealed significanthomologies with several proteins, including non-specific cross-reactiveantigen (NCA), biliary glycoprotein 1 (BG1), neural cell adhesionmolecule (NCAM), carcinoembryonic antigen (CEA), immunoglobulin alphachain constant region, the T cell receptor (TCR) alpha and beta chainvariable regions, and myelin associated glycoprotein (MAG). Lesserhomology is seen with myosin light chain kinase, ribulose biphosphatecarboxylase, adenovirus E1A 28K protein, pseudouridine synthetase, andxylulokinase. VCAM1 and 1b and the VCAM1 and 1b DNA sequences show nohomology with, and are distinct from, the previously described ELAM1(supra).

Importantly, NCA, BG1, NCAM, CEA, MAG, and TCR are members of theimmunoglobulin gene superfamily. (Williams and Barclay, 1988;Hunkapiller and Hood, 1989.) Members of this family are defined by thepresence of one or more regions homologous to the basic structural unitof immunoglobulin (Ig) molecules, the Ig homology unit. (Hunkapiller andHood, 1989.) These units are characterized by a primary amino acidsequence of about 70-110 residues in length, with an essentiallyinvariant disulfide bridge spanning 50-70 residues, and several otherrelatively conserved residues involved in establishing a tertiarystructure referred to as the “antibody fold”. These units may be furthersubdivided into three groups, i.e., V, C1, and C2 (Williams and Barclay,1988), or V, C, and H (Hunkapiller and Hood, 1989), based on variouscriteria, including intercysteine spacing, number of beta strands, andtype of conserved residues. When these criteria are applied to thepredicted primary sequence of VCAM1, the sequence can be divided intosix Ig units, designated domains 1-6, all of which fall into the C2 or Hsubset, each of about 100 amino acids in length. The invariant disulfidebridges of the six domains, referring to FIG. 3, occur between cysteines47 and 95 (domain 1), 137 and 195 (domain 2), 246 and 291 (domain 3),333 and 391 (domain 4), 442 and 487 (domain 5), and 531 and 576 (domain6).

As we stated above, VCAM1b has seven domains. We have designated theadditional domain as domain 3B. This domain is included in theadditional 276 nucleotides of VCAM1b that begin at nucleotide 1027 andend at nucleotide 1305 of FIG. 4. The DNA sequence encompassing domains1-3 is 72% homologous to the DNA sequence encompassing domains 3B-5. Atthe polypeptide level, there is significant homology between domains 1and 3B, 2 and 4, and 3 and 5, respectively. We present the domainstructures of VCAM1 and VCAM1b in FIGS. 5 and 6.

Messenger RNAs for VCAM1 and VCAM1b could arise by two mechanisms: Theycould represent alternately spliced forms of the same gene product, orthey could be the products of separate VCAM alleles. To help distinguishbetween these possibilities, we examined VCAM1 and mRNA from threeindividuals, at different time-points after cytokine induction. HUVECswere prepared from umbilical cords from three different individuals, thecord samples being labeled #1, #2 and #3. Each preparation was splitinto four separate flasks for treatment with TNF for 0 (untreated), 2.5,24, and 48 hours. Relative amounts of VCAM1 and VCAM1b mRNA weredetermined by Northern blotting and probing with syntheticoligonucleotides specific for each form. VCAM1b was clearly the majormRNA present in all three umbilical cord preparations. VCAM1 was presentin cords #1 and #3, most prominently at the 2.5 hour inductiontime-point, although in cord #3 VCAM1 was also present at 24 and 48hours. Cord #12 cells had little or no VCAM1 mRNA, although amounts ofVCAM1b mRNA were comparable to those in HUVECs from cords #1 and #3. Themechanism by which these two products arise is still unclear, althoughalternate splicing seems likely because the two mRNAs are identicalexcept for the deletion of one domain, at a point likely to be a splicejunction, judging by its position between domains (Hunkapiller and Hood,1989) and by the presence of the dinucleotide AG, typical of splicejunctions (Breathnach and Chambon, 1981). Furthermore, alternatesplicing is common among other members of the Ig gene superfamily towhich VCAM1 is most clearly related. (Hunkapiller and Hood, 1989.)

Functionally, differences between the two forms of VCAM1 appear to beminimal. Both forms, when expressed transiently in COS 7 cells, boundRAMOS cells, and this binding was completely inhibited by Moab 14B9,indicating that the same epitope is relevant to binding in each case.Furthermore, we have shown that this epitope is located within the firstthree domains, which are common to both forms (see Example VIII, supra).

EXAMPLE VIII Recombinant Soluble ELAM1 and VCAM1b

We constructed a vector expressing recombinant soluble ELAM1 (rsELAM1).We called this vector pSAB108. The rsELAM1 expressed by pSAB108 containsthe portion of the extracellular domain of ELAM1 encoded by the DNAsequence of FIG. 1 from nucleotide 141 to nucleotide 1790.

To construct pSAB108 we first created a DNA fragment which encoded anrsELAM1. We digested ELAM pCDM8 clone 6 with MluI and NotI. This yieldeda 3.8 kb DNA fragment including a DNA sequence encoding ELAM1. Wesubcloned this fragment into NotI-digested pNN11 that had been treatedwith calf intestinal alkaline phosphatase (described in Example III). Wecalled this vector pNNELAM1.

We used site specific mutagenesis to eliminate the transmembrane andintracellular regions of ELAM1. (Peden and Nathans, 1982; Kalderon etal., 1982; Oostra et al., 1983.) Accordingly, we digested a sample ofpNNELAM1 with EcoRI and isolated the large fragment. We linearizedanother sample of pNNELAM1 with ScaI. Then we synthesized anoligonucleotide having the sequence 5′ TGT GAA GCT CCC TAA ATT CCC. Whenthis sequence hybridizes to an ELAM1 antisense sequence it introduces astop codon and a BamHI restriction site into the ELAM1 DNA sequenceafter nucleotide number 1790. We created a heteroduplex using thesethree fragments according to the methods of Morinaga et al. (1984) andChang et al. (1984). We filled in the single stranded gaps with Klenowfragment and T4 ligase and used the mixture to transform E. coli MC1061.We screened the resulting colonies by checking for a BamHI site andselected mutagenized clones. Consequently on expression, thetransmembrane region of the polypeptide is eliminated and the C-terminalamino acid is proline. We called this plasmid pSAB100.

Then we digested pSAB100 with AatII and NcoI and isolated the 5.2 kbfragment. We also digested pNNELAM1 with these two enzymes and isolatedthe 1.4 kb fragment. NcoI cuts at nucleotide 927 of FIG. 1, about themiddle of the ELAM1 coding area. We ligated these two DNA fragments andcalled the plasmid pSAB108. We made this construction becausesite-directed mutagenesis sometimes causes mutations in other parts ofthe molecule and we wanted to avoid any such mutations in the codingregion or rsELAM1. We digested pSAB108 with NotI and isolated the 3.8 kbfragment. We ligated this fragment to a 7819 bp fragment of pBG341.jod,created as follows.

First we obtained pSV2-DHFR, ATCC 37146, from the American Type CultureCollection, Bethesda, Md. (USA). (Subramani et al., 1981.) We digestedthis with ApaI and EcoRI and isolated the 4420 bp fragment. Then, weproduced a synthetic double stranded DNA sequence having an ApaIoverhang, a DNA sequence encoding nucleotides +190 to +233 of the humangastrin gene (Sato et al., 1986, FIG. 4), an XhoI site, and an EcoRIoverhang. We ligated this oligonucleotide with the 4420 bp fragment ofpSV2-DHFR and called the resulting plasmid pDT4. We digested thisplasmid with AatII and XhoI and isolated the 4391 bp fragment.

Then we cleaved the Mullerian Inhibiting Substance expression vector pD1(Cate et al., 1986) with AatII and SalI and isolated the 5462 bpfragment. We ligated this fragment with the 4391 bp fragment of pDT4 tomake pJOD-10.

We digested pJOD-10 with HindIII and BstEII and isolated the largefragment which did not encode Mullerian Inhibiting Substance. Weblunt-ended the fragment ends, ligated SalI linkers to the ends andself-ligated the vector. This produced pJOD-s.

Then we digested pJOD-s with AatI and NotI and isolated the 6750 bpfragment. We ligated this to a 1100 bp NotI fragment from pBG341, whichwe created as follows.

We created pBG341 by replacing the SmaI site of pBG312 (Cate et al.,1986) with a NotI site. We linearized pBG312 with BqlII, blunt-ended thefragment by filling in with Klenow, and self-ligated it. We linearizedthis plasmid with BamHI and again blunt-ended and self-ligated it. Welinearized this plasmid with SmaI and ligated to the ends a NotI linkerhaving the sequence 5′ GCGGCGC. We called the resulting plasmid pBG341.

We digested pBG341 with AatII and NotI and isolated the 1100 bpfragment. We ligated this fragment to a 6750 bp fragment of pJOD-s. Wecalled the resulting plasmid pBG341.jod. This plasmid contains the SV40early and the adenovirus major late promoter. Genes inserted into theplasmid at the NotI site are transcribed from either of these promoters.

Then we linearized pBG341.jod with NotI and isolated the linear 7819 bpfragment. We ligated this fragment with the 3.8 kb fragment of PSAB108,which encoded rsELAM1, generating plasmid pSAB110.

We transfected CHO-DHFR cells by electroporation with plasmid pSAB110linearized with AatII. We performed electroporation with a Biorad® GenePulser at 270V and 960 μFD using 10⁷ cells/ml in 20 mM HEPES pH 7.05,137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄₁ and 6 mM dextrose with 20 μgplasmid and 200 μg sonicated salmon sperm DNA. Following transfection wecultured the cells in selective medium, alpha⁻ MEM containing 500nM,methotrexate and 10% dialyzed FCS. We picked colonies, plated them onto96-well cluster plates and detected rsELAM1-expressing cells using themonoclonal antibody BB11. After growing cells to confluence in completemedium containing 10% fetal calf serum (FCS) we maintained them inmedium containing 2% FCS in which the cells produced rsELAM1. Weharvested medium and replaced it with fresh 2% serum every three or fourdays.

We isolated rsELAM1 from this conditioned medium to at least 95% purity.This involved concentrating the medium and incubating it overnight withMoab BB11 (Example V) covalently coupled to Protein A sepharose.(Schneider et al., 1982.) Then we washed this resin with PBS to removeunbound protein, eluted bound material with 0.1 M glycine, pH 2.7,neutralized the eluate with sodium phosphate and dialyzed it againstPBS. We further purified the rsELAM1 by chromatography with Protein Asepharose in PBS.

Using the following assay, we demonstrated that we had produced rsELAM1.To a 6 cm diameter Petri dish of bacteriologic plastic (e.g., Falcon#1007®) we added 2.5 ml of 50 mM Tris buffer, pH 9.5. To this we added10 μg of pure rsELAM1. We incubated the plate for 60 minutes at roomtemperature to allow the rsELAM1 to bind to the plate. Then we aspiratedthe medium and replaced it with PBS containing 10 mg/ml bovine serumalbumin. We incubated the plates overnight at 4° in this solution toblock remaining protein binding sites on the plates. We warmed theplates to room temperature, washed them with medium containing 10% fetalcalf serum, and incubated them with 2 ml of cells (2×10⁶ ml⁻¹) for 20minutes. We aspirated the medium and washed the plates twice with 3 mleach of medium (RPMI 1640 with 10% serum). Then we examined the platesby microscopy.

We found that cells which bind ELAM1, such as HL-60 cells, bind torsELAM1-coated plates, while cells which do not bind to ELAM1, e.g., theB cell line RAMOS, do not bind to these plates.

In addition, we found that the specific Moab BB11 blocks the binding ofHL-60 cells to rsELAM1 coated plates. Together, these results showfirst, that we have produced rsELAM1, and, second, that like ELAM1,rsELAM1 possesses the ability to bind to leukocytes.

We also constructed a vector expressing recombinant soluble VCAM1b(rsVCAM1b). This vector was named pBN1006, and the rsVCAM1b expressed bypBN1006 contains the portion of the extracellular domain of VCAM1bencoded by the DNA sequence shown in FIG. 4 from nucleotide 107 tonucleotide 2193.

In order to generate a cell line capable of constitutively expressingfull length soluble VCAM1b, we first created a vector derived frompJOD-s having a unique NotI site downstream from the adenovirus majorlate promoter, so that NotI fragments could be inserted into theexpression vector. pJOD-s was linearized by NotI cleavage of the plasmidDNA. The protruding 5′ termini were blunt-ended using Mung-bean nucleaseand the linearized DNA fragment was purified by low melting temperatureagarose (LMA) gel electrophoresis. The DNA fragment was religated usingT4 DNA ligase. The ligated molecules were then transformed into E. coliJA221 (ATCC accession no. 33875). Colonies were screened for the absenceof a NotI site. The resulting vector was designated as pJOD-s deltaNot1. pJOD-s delta Not1 was linearized using SalI and the 5′ terminiwere dephosphorylated using calf intestine alkaline phosphatase. Thelinearized DNA fragment was purified by LMA gel electrophoresis andligated in the presence of phosphorylated oligonucleotide ACE 175 (5′pTCGACGCGGCCGCG). The ligation mixture was transformed into E. coliJA221 and colonies were screened for the presence of a NotI site. Thecorrect plasmid was named pMDR901.

Soluble VCAM1b was obtained by truncating VCAM1b clone 1E11 atnucleotide 2193 by digestion with AluI, thus eliminating thetransmembrane and intracellular portion as well as the 3′ untranslatedregion. A stop codon-NotI linker was added, and the insert was religatedinto pCDM8. The insert was excised from PCDM8 with NotI and ligated intopMDR901 at the NotI site. This construct, designated pBN1006, encodesfull length soluble VCAM1b, having amino acids 1-698 as shown in FIG. 4.

Using materials and methods already described, we have also constructedplasmids expressing truncated forms of the rsELAM1 and rsVCAM1bmolecules described above. These truncated forms, comprising the aminoacid sequences of one or more of the particular domains of theextracellular regions of ELAM1 and VCAM1b, were used to investigatewhich domain or domains are involved most directly in cell-to-celladhesion. Our initial experiments have investigated the domains of ELAM1and VCAM1 and 1b that are recognized by antibodies against thosemolecules, i.e., antibodies BB11 and 4B9, respectively.

A soluble ELAM1 construct designated CH101 was prepared comprising thelectin-like domain of ELAM1. Referring to FIG. 1, CH101 was theexpression product of a cDNA sequence including nucleotides 1-557(coding for amino acids 1 through 139 of ELAM1) and a stop codon.Another soluble construct designated CH102 was prepared comprising thelectin-like domain and the EGF-like domain of ELAM1. Referring to FIG.1, CH102 was the expression product of a cDNA sequence that includednucleotides 1-671 (coding for amino acids 1 through 177 of ELAM1) and astop codon. The soluble ELAM1 construct CH102 was found toimmunoprecipitate the anti-ELAM1 monoclonal antibody, BB11.

The following soluble VCAM1 and 1b constructs were similarly prepared:

(A) domain 1 (nucleotides 1-430 of FIG. 3, coding for amino acids1-108);

(B) domain 1+domain 2 (nucleotides 1-757 of FIG. 3, coding for aminoacids 1-217);

(C) domain 1+domain 2+domain 3 (nucleotides 1-1036 of FIG. 3, coding foramino acids 1-310);

(D) domain 1+domain 2+domain 3 (from a hybrid of VCAM1 and VCAM1b cDNA,coding for amino acids 1-317 as depicted in FIG. 4);

(E) full length soluble VCAM1 (nucleotides 1-1924 of FIG. 3, coding formamino acids 1-606); and

(F) full length soluble VCAM1b (nucleotides 1-2193 of FIG. 4, coding foramino acids 1-698).

Of the foregoing VCAM1 constructs, B, C, D, E and F (but not A) wereimmunoprecipitated with the anti-VCAM1 antibody 4B9. Constructs B. D, Eand F were also found to produce protein functional for cell adhesion.Conditioned media containing protein encoded by constructs B, D, E and Fwere concentrated, passed over an immunoaffinity column of immobilized4B9 antibody, and the bound protein eluted and neutralized as describedfor rsELAM1. The eluted proteins were immobilized on plastic asdescribed for rsELAM1 and found to support specific adhesion of RAMOSand JURKAT cells. These results indicate that the first two domains ofVCAM1 are sufficient to support adhesion of certain VLA4-expressinghuman lymphoid cell lines.

EXAMPLE IX Isolation of the ELAM1 and VCAM1 Promoter

We have isolated and characterized genomic clones for the ELAM1 andVCAM1 genes. We isolated the ELAM1 clones as follows:

We selected as probes either the entire ELAM pCDM8 clone 6 insert or a400 base pair fragment from its 5′ end. We labeled these molecules with³²P by random priming. Then we screened a human genomic EMBL3 librarywith the ELAM cDNA probes. We isolated and characterized a genomic ELAM1clone from the library and designated it EL1-07. It includesapproximately 15 kb of 5′ flanking sequence including thetranscriptional promoter for ELAM1 and approximately 100 base pairs ofcoding sequence at the 5′ end of the gene. Current knowledge suggeststhat the relevant control sequences for induction will be includedwithin the DNA sequence represented by this phage clone. (Leonardo andBaltimore, 1989.) We sequenced a region including 840 bp of 5′ flankingsequence and 720 bp of the 5′ end of the ELAM1 gene, including the firsttwo exons, the first intron and part of the second intron. We presentthis sequence in FIG. 7. The 5′ flanking region displays a classicalpromoter structure including TATAAA and CAAT sequences. It also containsthe sequence GGGGATTTCC about 95 base pairs upstream from the presumedstart of transcription. This sequence is an NF-κB binding sequenceidentical to that found in the human κ immunoglobulin (Ig) geneenhancer. NF-κB is an inducible DNA binding protein known or suspectedto stimulate transcription of a number of genes relevant to inflammationand the immune response (such as the immunoglobulins, the interleukin-2receptor, and β-interferon, among others). It can be activated by TNF,IL-1, and LPS, the same inducers known to stimulate production of ELAM1,VCAM1, and ICAM1. (Lenardo and Baltimore, 1989; Osborn et al., 1989.) Wehave demonstrated that NF-κB DNA binding activity is stimulated inendothelial cells by IL-1 and TNF, and we are currently engaged indefining minimal DNA sequences necessary for inducible transcriptionfrom the ELAM1 promoter, by transfection of promoter/reporter geneconstructs into endothelial and other cell types.

We deposited clone EL1-07 under the Budapest Treaty with In VitroInternational, Inc., Linthicum, Md. (USA) on Dec. 7, 1989. It isidentified as:

-   -   EL1-07    -   Accession Number IVI-10218.

We also isolated an EMBL3 genomic clone representing the VCAM1 gene byprobing the previously mentioned EMBL3 human genomic library with a³²P-labeled 30 base oligomer probe homologous to the 5′ end of the VCAM1cDNA. We designated this clone VC1-16 and deposited it under theBudapest Treaty with In Vitro International, Inc., Linthicum, Md. (USA)on Dec. 7, 1989. It is identified as:

-   -   VC1-16    -   Accession Number IVI-10217.        We sequenced a region including approximately 300 bp of 5′        flanking sequence and 900 bp of the 5′ end of the VCAM1 gene,        including the first exon, the first intron, and part of the        second exon. We present this sequence in FIG. 8. The 5′ flanking        region has a classical TATAAA sequence, and two NF-κB consensus        sequences: AGGGATTTCC on the sense strand from about −63 to −54        from the start of transcription, and GGGGAAACCC on the reverse        complement strand from about −69 to −78. This sequence will be        used for studies analogous to those proposed for the ELAM1        promoter sequence.

EXAMPLE X Antibodies Recognizing CDX

We isolated CDX, a MILA involved in ELAM1-mediated adhesion. As a firststep, we prepared monoclonal antibodies that recognized an antigen onthe leukocyte cell surface and that interfered withleukocyte-endothelial cell binding. In order to assure that the antigenthat these monoclonals recognized was involved in ELAM1-mediatedadhesion, we tested the monoclonals in systems in which ELAM1-mediatedbinding was the exclusive cell-cell binding pathway.

1. Preparation and Analysis of Monoclonal Antibodies Against CDX

a. Adhesion Assay

To identify Moabs that inhibit leukocyte-endothelial cell binding, wedeveloped an improved assay to detect endothelial cell-leukocyteadhesion. We performed this assay using HL-60 cells and HUVECs. Itshould be clear that one can perform such an assay using any cell linethat expresses a MILA and with any cell line that expresses an ELAM. In48-well tissue-culture plates we grew HUVECs to confluence (8×10⁴cells/well). We washed the cells once with RPMI/1% FCS and added 0.5 mlRPMI/1% FCS with 13 U/ml of IL-1β to each well (except the controlwells). We incubated these cells for 4 hours at 37° C. Just before use,we washed them once with RPMI/1% FCS. The HL-60 cells we used in theassay had been labeled overnight with 1 μCi/ml of ³⁵S-methionine. Wewashed these cells once and then resuspended them in RPMI/1% FCS at5×10⁶ cells/ml. We took 100 μl of the HL-60 cells and incubated them for30 min at 0° C. with 50 μl of Moab (1 μg/ml). Then we added the 150 μlto each well of HUVECs. We allowed the cells to bind for 10 min at 20°C. and then washed the wells gently once with RPMI/1% FCS. We filled thewells with RPMI/1% FCS, sealed the plates, inverted them, andcentrifuged them for 2 min at 500×g. We removed the media and washed thewells two more times with PBS⁼. (PBS⁼ is PBS without Ca⁺⁺ and withoutMg⁺⁺.) We determined the number of HL-60 cells bound to the HUVECs bysolubilizing the cells in each well with 200 μl of 0.2N NaOH/1% SDS,adding 4.5 ml of scintillant (Ready Protein, Beckman), and counting witha scintillation counter.

b. Preparation of Hybridomas

To make monoclonal antibodies against CDX we prepared hybridomas in thefollowing manner. We injected BALB C mice with whole, live HL-60 cells.Initially, each mouse received 2×10⁷ cells in PBS⁼ intraperitoneally(IP). We injected complete Freund's adjuvant intraperitoneally at adifferent site 2-24 hours later. We boosted the mice with 2×10⁷ cells IPevery second week for six weeks. Four days before fusing we injected themice intravenously with 5×10⁶ cells and IP with 5×10⁶ cells.

We tested immune serum from these animals for the ability to inhibitbinding of the HL-60 cells to IL-1β stimulated HUVECs by the adhesionassay described above. The immune serum tested positive after the thirdboost and we proceeded to produce hybridomas from the spleen cells ofthe immunized animals. We performed fusion of spleen cells and myelomacells in a manner standard to the art. (See, Goding, 1983.)

Using the adhesion assay we described above, we screened the hybridomasfor those producing monoclonal antibodies that inhibited the binding ofHL-60 cells to IL-1β-induced HUVECs. In this way we identifiedhybridomas that produced monoclonal antibodies that recognized CDX. Weused five of these hybridomas to produce ascites fluid. We deposited oneof them, designated SGB₃B₄, under the Budapest Treaty with In VitroInternational, Inc., Linthicum, Md. (USA) on Apr. 25, 1989. It isidentified as:

-   -   SGB₃B₄    -   Accession number: IvI-10205

c. FACS Analysis

To identify to which cell types our monoclonals bound, we performed FACSanalysis. This involved taking 2×10⁵ cells, washing them one time withPBS⁼, and then blocking Fc receptors by incubation in 25 μl of RPMI, 1%FCS, 0.1 mg/ml human IgG, and 0.1% sodium azide for 10 min at 0° C. Wethen added antibody (25 μl at 1 μg/ml) and incubated the cells 30 min at0° C. We centrifuged the cells at 250×g for 5 min, washed them two timeswith Buffer A (PBS®, 5% FCS, 0.1% azide) and resuspended them in 25 μlBuffer A containing 0.1 mg/ml human IgG. We added fluorescein-conjugatedanti-mouse IgG (25 μl at 5 μg/ml in Buffer A (Cappel)) and incubated themixture 30 min at 0° C. We centrifuged the cells, washed them once withBuffer A, and resuspended them in 250 μl Buffer A. Then we analyzed themon a Becton-Dickinson FACStar Cell Sorter.

We performed cell binding studies with the ELAM1-expressing COS cellsessentially as described for the HL-60 cell-HUVEC adhesion assay.

2. Demonstration That Hybridoma SGB₃B Produced Monoclonal AntibodiesThat Recognize CDX

We have developed several lines of evidence that demonstrate thatmonoclonals from hybridoma SGB B specifically recognize a MILA involvedin ELAM1-mediated binding, specifically, CDX.

First, the α-CDX antibodies should inhibit binding of cells expressingCDX to ELAM1-expressing cells. Using the adhesion assay, we showed thatthese monoclonals do indeed inhibit the binding of HL-60 cells and PMNsto IL-1β-induced HUVECs and ELAM1-expressing COS 7 cells. In thepresence of 60.3, a monoclonal antibody against the β₂ integrin chain,the only binding pathway for HL-60 cells and PMNs that is utilized inELAM1-expressing COS 7 cells is ELAM1 itself. Therefore, antibodyinhibition of cell-cell adhesion in this system must be through theELAM1 pathway via CDX.

Second, α-CDX monoclonals should recognize those cells that bind toELAM1-expressing cells in an adhesion assay, but should not recognizethose cells that do not bind to ELAM1 in this assay. Using FACSanalysis, we determined the binding pattern of our Moabs. Thesemonoclonals bound to the following cell types: HL-60, U937, HT-29,THP-1, SW620, SW948, SW1417, monocytes, eosinophils, and PMNs. They didnot bind to these cells: RAJI, DAUDI, RAMOS, HeLa, or JY. (We isolatedthe non-transformed cells by fractionating peripheral blood leukocytes.)This binding pattern precisely parallels the binding of these cells toELAM1-expressing COS 7 cells and to rsELAM1-coated plates.

Third, α-CDX monoclonals should exhibit a different recognition patternthan monoclonals against other leukocyte cell-surface antigens, such asLFA-1, LFA-3, CD44, ICAM1 and CD4. In fact, no other monoclonal of whichwe are aware exhibits the same cell-recognition pattern as ourantibodies.

Fourth, and most convincing, using these MoAbs we cloned a gene that canconfer ELAM1 binding activity in cells that otherwise do not bind toELAM1.

In sum, it is apparent that the monoclonals produced by hybridomaSGB₃B₄, and by other hybridomas we isolated, recognize CDX.Consequently, we used these monoclonals to isolate CDX itself.

EXAMPLE XI Isolation of CDX

1. Iodination of HL-60 Cell Surface Proteins

We washed 1×10⁷ HL-60 cells three times with PBS═, resuspended them in0.5 ml PBS⁼ and added them to a tube coated with 50 μg1,3,4,6-tetrachloro-3α,6α-diphenylglycouril (Sigma Chemical Co.). Tothis we added 1 mCi of ¹²⁵I. We incubated the mixture for 30 min at 0°C. We transferred labeled cells to a tube containing 10 ml of RPMI/10%FCS and centrifuged them at 1000×g for 5 min. Then we washed them firstwith another 10 ml of RPMI/10% FCS and second with 2 ml of PBS®.(Alternatively, we have labeled the cells metabolically with³⁵S-methionine or 5-cysteine.) We lysed the cells by addition of 1.0 mlPBS⁼ containing 1% NP40, 2 mM PMSF, 1 mM EDTA, soybean Trypsin inhibitor(50 mg/ml), and Leupeptin (1 mM) (Sigma Chemical Co.). Then we incubatedthem for 30 min at 0° C. We centrifuged the lysate for 10 min at10,000×g to remove particulate matter. We precleared the supernatantcontaining labeled solubilized membrane proteins with 10 μg of rabbitanti-mouse IgM (Jackson Immuno-Research Labs) and 50 μl of Protein Asepharose (Zymed, 2 mg Protein A/ml) for 2 hours at 0° C. We stored thelysate at 4° C.

2. Immunoprecipitation of CDX

We purified CDX away from the other labeled proteins using the Moabs toimmunoprecipitate it. We performed the immunoprecipitation as follows:

We incubated precleared lysate (50-100 μl) with 10λ of ARX beads for 2hours at 4° C. We washed the sepharose four times with 2 ml PBS⁼containing 0.75% NP40, 0.2% DOC, and 1 mM EDTA. Then we resuspended theARX beads in non-reducing SDS sample buffer. We heated the sample for 10min at 85° C. and removed the supernatant. To this we added B-ME to 5%,heated for 5 min, and separated the molecules on a 10% SDSpolyacrylamide gel. We dried the gel and autoradiographed it.

CDX appeared on the autoradiograph as a single, diffuse band withmolecular weight of approximately 150 kD.

EXAMPLE XII Isolation and Characterization of Clone 7.2 and Clone 1

We prepared two cDNA libraries in the pCDM8 vector from two types ofCDX-expressing cells, HL-60 cells and U937 cells. We isolated the mRNAfrom these cells and reverse-transcribed it into cDNA using techniqueswell known to the art. (Gubler and Hoffman, 1983.) Using standardprocedures, we ligated double stranded cDNA to a NotI-BstXIlinker/adaptor having the following sequence: 5′ GCG GCC GCT TTA GAG CACA 3′ 3′ CGC CGG CGA AAT CTC       5′We then size-selected the cDNA on a 4.2 ml 5-20% potassium acetategradient, 2 mM EDTA, 1 μg/ml ethidium bromide, in a BECKMAN SW60 Rotorfor 3 hours at 50,000 rpm at 22° C. according to the protocols of BrianSeed. (See also Maniatis, 1982, p. 278.) We pooled the cDNA fragments ofgreater than 500 base pairs. Then we prepared the vector, PCDM8 (a giftfrom Brian Seed). We digested this plasmid with BstXI. To remove the 400base pair stuffer fragment we centrifuged the mixture on a potassiumacetate gradient, as above, and isolated the large fragment. We furtherpurified this fragment by agarose gel electrophoresis, and then ligatedthe cDNA to the vector.

We then prepared an enriched cDNA library by first creating a³²P-labeled cDNA probe from 1 microgram of HL-60 poly A+ mRNA, thensubtracting non-CDX related cDNA sequences from the probe by hybridizingwith 30 micrograms of poly A+ mRNA from HeLa cells, which do not expressCDX. (See, Davis, 1986.) We used the subtracted probe to screen thepCDM8 cDNA library and thus created an enriched sublibrary from HL-60cells in E. coli MC1061 P3. We grew about 2100 clones in twenty-two96-well plates. A U937 enriched sublibrary was prepared in a similarmanner, and 1400 clones were obtained.

We divided the colonies from our HL-60 enriched library into 22 poolsfor transfection of COS 7 cells by spheroplast fusion. (Sandri-Goldin etal. 1981.) We assayed transfected COS 7 cells for ELAM1-binding activityby panning with α-CDX monoclonal antibodies from hybridoma SGC2E₅ (anantibody similar in function to SGB₃B₄) according to the method of Seedand Aruffo (1987). (See also Aruffo and Seed, 1987 and Wysocki and Sato,1978). Pool #7 assayed positive, yielding two clones with a 2.1 kb cDNAinsert. These were designated clones 7.1 and 7.2.

We obtained the DNA sequence of clone 7.2 by the Maxam and Gilberttechnique (Maxim and Gilbert, 1980) from CDX pCDM8 clone 7.2 and from aportion of the 7.2 insert subcloned into the sequencing vector, pNN11.The latter plasmid was designated pSQ219. The DNA sequence obtained isset forth in FIG. 9.

We deposited a culture containing the plasmid CDX pCDM8 clone 7.2 underthe Budapest Treaty with In Vitro International, Inc., 611 P. HammondsFerry Rd., Linthicum, Md. 21090 (USA) on Apr. 26, 1990. The deposit isidentified as:

-   -   CDX pCDM8/E. coli MC1061 P3    -   Accession Number IVI-10242

We also performed a Northern blot on mRNA from HL-60 cells and probed itwith clone 7.2. Clone 7.2 hybridized to three mRNA species, twoprominent bands at 6.0 kb and 2.4 kb and another band at 3.0 kb. Clone7.2, a cDNA of 2.1 kb, is not large enough to be a full length cDNA fromthe 3.0 kb and 6.0 kb species. Therefore, in order to identify DNAsequences for these messages, we probed the enriched cDNA sublibraryfrom both U937 and HL-60 cells with an oligonucleotide derived fromclone 7.2. We isolated several long inserts from the HL-60 library,transfected them into COS 7 cells, and selected clones that bound toELAM1 and α-CDX. In this way we identified a 2.9 kb insert that couldhave come from the 3.0 kb message. We called it CDX clone 1.

We determined the DNA sequence of CDX clone 1 by the Maxam and Gilberttechnique. The DNA sequence obtained is set forth in FIG. 10.

We deposited a culture containing the plasmid CDX clone 1 under theBudapest Treaty with In Vitro International, Inc., 611 P. Hammonds FerryRd., Linthicum, Md. 21090 (USA) on Oct. 11, 1990. The deposit isidentified as:

-   -   CDX clone 1 pCDM8/E. coli MC1061 P3    -   Accession Number IVI-10255.

We transfected clone 7.2 and clone 1 into COS 7 and CHO cells. At 48hours after transfection these cells expressed a glycoprotein on theircell surfaces to which fluorescently labelled α-CDX antibodies bound, asassayed by FACS. These cell surface proteins could be labeled with ¹²⁵Iand immunoprecipitated with α-CDX Moabs. We designated the proteinisolated from COS 7 cells, Pseudo-X and from CHO cells, Pseudo-X₂. OnSDS polyacrylamide gels, Pseudo-X and Pseudo-X₂ were approximately 130kD and 140 kD, respectively.

The transfected COS cells also formed rosettes around Sepharose beadscoated with recombinant soluble ELAM1 (rsELAM1); and the rosetting wascation dependent and was inhibited by both BB11 (anti-ELAM1 antibody)and α-CDX. COS cells and CHO cells transfected with PCDM8 alone (withoutthe inserted clone) did not rosette rsELAM1 beads. Also, the COS and CHOcells transfected with clone 7.2 did not rosette to beads coated withbovine serum albumin.

We further characterized clone 7.2 and clone 1 by DNA sequence analysisand enzyme assays. Clone 1 encodes a polypeptide of 530 amino acids(encoded by nucleotides. 174-1763 of FIG. 2). Clone 7.2 encodes a405-amino acid polypeptide (encoded by nucleotides 66-1280 in FIG. 1).Using UWGCG Sequence Analysis Software Package (version 6.1, August1989), we searched the NBRF Protein database (release 23, December 1989)using the program FASTA for homology to other proteins. We also searchedGenBank (release 63, March 1990) and EMBL (release 19, May 1989) usingTFASTA. In these searches we found short regions (e.g., about 23 aminoacids) of homology to certain viral envelope proteins including Herpessimplex virus type 1, Dengue virus, yellow fever and other flaviviruses.In general the homology to known proteins was low, and we conclude thatthe polypeptides are novel.

The portion of the nucleotide sequence of clone 7.2 from nucleotide 9 tonucleotide 2162 (FIG. 9) is identical to the portion of the sequence ofclone 1 from nucleotide 492 to nucleotide 2645 (FIG. 10). The firstmethionine of protein 7.2 corresponds to the methionine at amino acid126 of protein 1. One explanation of this homology is that the twoinserts represent different transcripts from the same DNA segment.

As we stated earlier, these clones do not code for CDX, Pseudo-X orPseudo-X₂—the polypeptides they encode are not the correct, size.Rather, the evidence strongly supports the conclusion that clone 7.2 andclone 1 encode 1,3-fucosyl transferases that glycosylate other proteins,such as CDX, Pseudo-X and Pseudo-X₂, in a way that makes them “visible”(i.e., recognized by or able to bind to) ELAM1 or α-CDX. First, the DNAsequences of clone 1 and clone 7.2 share several structural featureswith the DNA sequences of known glycosyl transferases. For example,genes encoding known glycosyl transferases commonly have consecutivemethionine start sites and are capable of producing more than one mRNAtranscript. As mentioned above, we have identified three mRNAtranscripts that hybridize to clone 7.2, and clone 1 contains two codonsthat can serve as transcription start signals. Also, like known glycosyltransferases, the clones have multiple SP1 enhancer sites. Thenucleotide sequences for these sites are GGGCGG or CCGCCC; clone 1 hasfive such sites. Also, like known glycosyl transferases, clones 7.2 and1 are rich in guanine (G) and cytosine (C). For example, clone 1 is 75%GC rich in the 5′ region of the gene and 60% GC rich in the 3′ region ofthe gene. Glycosyl transferases in addition are typically class IImembrane proteins, in which the membrane-spanning domain is near theamino terminus and the extracellular portion is near the carboxyterminus. Clone 1 and clone 7.2 encode a polypeptide having ahydrophobic region near the amino terminus. Glycosyl transferases alsotend to have molecular weights between 40 kD to 60 kD; clone 1 encodes apolypeptide of about 59 kD and clone 7.2 encodes a polypeptide of about46 kD. Finally, known glycosyl transferases usually have one to threeN-glycosylation sites; clone 1 and clone 7.2 both encode two such sites.

Second, enzyme assays performed on extracts from CHO cells transfectedwith clone 7.2 revealed the presence of fucosyl transferases notexpressed in untransformed cells. The assays tested the ability of theenzyme to link radioactively labelled fucose to an acceptor molecule. Weperformed the assays as follows.

We prepared assay samples containing 10 μl enzyme, 8 μl cocktail and 2μl 10× acceptor. We prepared the enzyme by isolating about 1.5 millionCHO cells transfected with clone 7.2 and lysing them by sonication for15 seconds in 150 μl ice-cold 1% Triton X-100 in water. The cocktailcontained 75 μM ¹⁴C-GDP fucose, 100 mM ATP, 500 mM L-fucose, 1 M MnCl₂and 1 M cacodylate at pH 6.2. 10× acceptor contained, variously, 200 mMLacNAc, Lac-N-biose, or lactose, 250 mM phenyl-β-D-galactoside, or 50 mM2′-fucosyllactose. We incubated the assay samples for 1 hour at 37° C.We stopped the reaction by addition of 20 μl ethanol. We diluted thesample with 560 μl water and centrifuged in an EPPENDORF centrifuge for5 minutes at high speed.

We had prepared a DOWEX 1×2-400 column (Sigma Chemical Co.) to separatethe unconverted ¹⁴C fucose-GDP from the converted. We loaded the matrixinto a large column and washed it with 10 volumes of 1N NaOH, followedby 5 volumes of water, followed by 10 volumes of 5% concentrated formicacid. Then we repeated this wash cycle. We used this material to createsmall columns of 0.4 ml. We prepared the small columns for use bywashing them with 10 volumes of water.

We loaded 200 μl of the sample onto the small column, collected theeluate, rinsed with 2 ml water and collected it into the eluate. Wedetermined the radioactivity of this eluate by scintillation counting.

The results of this assay demonstrated that the induced enzyme is a1,3-fucosyl transferase. (See Table 1.) The enzyme linked fucose toLacNAc, 2′-fucosyllactose and lactose, acceptors having GlcNAc orglucose moieties with free 3′ hydroxyls. It did not link fucose toLacNBiose, whose GlcNAc moiety does not have a free 3′ hydroxyl, orphenyl-β-D-galactoside, the negative control acceptor. Control samplesfrom untransfected cells showed only insignificant linking of fucose tothese acceptors. TABLE 1 Efficiency of Fucosylation picomoles Acceptormg Total protein · hr LacNAc 1110 Lac-N-Biose 76 2′-Fucosyllactose 151Lactose 290 PhβDgal Not detectable[The enzyme was freshly produced from transfected CHO cells.]

Therefore, both genetic and enzymatic evidence indicate that clone 7.2and clone 1 encode 1,3-fucosyl transferases.

EXAMPLE XIII Antibodies Recognizing MILAs For VCAM1

Polyclonal antisera were obtained from three mice that had beenimmunized with whole JURKAT cells. The serum from one mouse completelyinhibited both RAMOS and JURKAT binding to 4 hour-induced HUVECs at roomtemperature. The sera from the two other mice completely inhibited RAMOSbut only partially inhibited JURKAT binding under the same conditions.These data indicate that RAMOS and JURKAT share a MILA, and that JURKATexhibits at least one other MILA not shared by RAMOS.

To prepare Moabs to lymphocyte MILAs, we immunized mice against wholelive RAMOS and JURKAT cells and performed fusion of spleen cells fromJURKAT-immunized mice and myeloma cells in the manner described inExample VIII, above. We are screening the resulting hybridomas by themethod described in Example VII, which we used successfully to obtainmonoclonal antibodies to CDX. To date we have screened the conditionedmedium from about 260 hybridomas for inhibition of RAMOS adhesion toHUVECs treated with TNF for 24 hours. About 25 hybridomas have shownconsistent partial inhibition of adhesion, and these are currently beingsubcloned for re-screening. Such antibodies may be used to both isolateand clone lymphocyte MILAs.

EXAMPLE XIV Evidence that VLA4 is a VCAM1 Ligand

We and other colleagues have performed several studies that demonstratethat VLA4 is a VCAM1 ligand and that VLA4 has separate binding sites forVCAM1 and fibronectin.

First, we showed that monoclonal antibodies against the subunits of VLA4inhibited the attachment of VLA4-expressing cells to activated HUVECsand to COS cells transfected with VCAM1. VLA4 is composed of thesubunits β₁ and α⁴. (Hemler, 1988.) We found that a monoclonal antibodyagainst β₁, designated B1E11, and goat anti-β₁ heteroantiserumcompletely inhibited the adhesion of RAMOS cells to activated HUVECs andtransfected COS cells. A control antibody did not inhibit adhesion.Furthermore, a monoclonal antibody against the α⁴ subunit, designatedHP2/1, also blocked attachment of RAMOS to these cells. Similarly, theseantibodies inhibited the attachment of the VLA4-expressing Tlymphoblastoid cell line HPB-ALL.

Next, we showed that transfecting cells that do not ordinarily expressVLA4 with α⁴ enabled them to bind to VCAM1-expressing cells. Wetransfected two sets of K-562 erythroleukemic cells. One set wastransfected with a cDNA coding for α⁴. (Takada et al., 1989.) The otherwas transfected with α², which is not part of VLA4. (Takada and Hemler,1989.) We showed that K-562 cells transfected with α⁴ were now able tobind with a monolayer of VCAM1-transfected COS cells or TNF-activatedHUVECs, but parent K-562 cells and K-562 α²-transfected cells were not.In addition, monoclonal antibodies against α⁴ or β₁ abolished theadhesion of α⁴-transfected K-562 cells (that normally express thesubunit) to these VCAM1-expressing cells.

Recent studies have shown that VLA4 mediates cell attachment to humanplasma fibronectin (FN) through the FN CS-1 site. (Wayner et al., 1989.)We have shown that the VLA4 binding site for VCAM1 is different than itsbinding site for FN. First, we found that preincubation of RAMOS cellsor α⁴-transfected K-652 cells with FN-40 (a soluble FN fragment)inhibited their binding with FN-40, but not with VCAM1-transfected COScells or TNFα activated HUVECs. Second, we found that a monoclonalagainst VLA4, HP1/3, inhibited the binding of these cells to transfectedCOS cells or activated HUVECs, but not to FN-40.

EXAMPLE XV Inhibitor Screening

One can use ELAMs and their ligands in three basic adhesion assays toscreen for potential inhibitors of adhesion, such as synthetic organicchemicals, natural fermentation products, peptides, etc.:

1. Cell-Cell Adhesion Assays

A first assay would test the ability of molecules to inhibit cell-celladhesion. One could perform this assay in 96-well microtiter plates.First, one creates a cell line that stably expresses an ELAM, forexample, as described in Example V. Then one plates out these cells andadds HL-60 cells. Inhibitors are identified by their ability to inhibitHL-60 binding to the ELAM-expressing cells. One would perform an assayexactly as described for screening for monoclonal antibodies to the ELAMligand.

2. Cell-Adhesion Protein Assays

A second assay would test the ability of a small molecule to inhibitcell binding to ELAM itself. We have developed such an assay withrsELAM1 which works in 96 well microtiter plates. These plates, made ofbacteriologic plastic (e.g. Linbro/Titertek #76-232-05®), are incubatedwith 0.5 μg per well of rsELAM1 in 50 μl of 15 mM sodium carbonate/35 mMsodium bicarbonate, pH 9.2, overnight at 40. The plates are then blockedfor one hour at room temperature with PBS containing 10 mg/ml of bovineserum albumin, and then adhesion assays performed as described inExample VIII using, e.g., HL-60 cells, 2×10⁶/ml, 50 μl per well. Underthese conditions HL-60 cells bind well to rsELAM1, providing aconvenient microassay for screening. One would identify inhibitors bytheir ability to inhibit HL-60 binding to the plate. Alternatively, onecould use an ELAM ligand in this assay, using as the probe a cell linethat stably expresses an ELAM.

Another alternative assay in this category would examine the binding ofa soluble ELAM or ELAM ligand to monolayers of cells stably expressingan ELAM ligand or ELAM, respectively. The soluble molecule would belabeled with a reporter group (e.g., radioactivity, fluorescent probe,enzyme, etc.)

3. Adhesion Protein-Adhesion Protein Assays

This assay tests the ability of a small molecule to inhibit the bindingof an ELAM to its ligand. One of the two molecules in soluble form,e.g., a soluble ELAM, is immobilized in the wells of a 96-wellmicrotiter plate, and adhesion is measured by binding of the othermember of the pair, e.g., an ELAM ligand labeled with a reporter group.

In each of these three assays, one detects inhibitors by their abilityto inhibit adhesion.

EXAMPLE XVI VCAM1/Immunoglobulin Construct

We have prepared a DNA sequence which, on expression, produces anrsVCAM1/immunoglobulin fusion protein. The DNA sequence contains, from5′ to 3′, VCAM1 domains 1-3 and the constant region of an IgG heavychain gene.

We produced a DNA fragment containing the VCAM1 domains 1-3 throughnucleotide 1035 of FIG. 3 by polymerase chain reaction (PCR). (Sambrooket al., 1989) The 3′-5′ primer had the sequence 5′ GA GCT CGA GGC CGCACC ATG CCT GGG AAG ATG. It is complementary to nucleotides 100-114 inFIG. 3 and contains the VCAM1 initiation codon and recognition sites forXhoI and NotI. The 5′-3′ primer had the sequence 5′ CT AGC TAG CGC GTTTTA CTT CAC. It is complementary to nucleotides 1016-1035 in FIG. 3, atthe end of domain 3, and contains an NheI recognition site. We usedthese primers to amplify a segment from a plasmid containing VCAM1coding region of AM pcDM8 clone 41. The product of this process was aDNA sequence encoding VCAM1 domains 1-3. We digested this DNA fragmentwith XhoI and NheI and inserted it into pAB53, which we made as follows.

We digested pJOD-s (Example VIII) with SalI and inserted a cDNA sequenceencoding human rsCD4. We called this plasmid pJOD-rsT4. We partiallydigested pJOD-rsT4 with PvuII and SphI to delete the fragment containingthe two SV40 enhancer repeats in the SV40 promoter which controltranscription of the DHFR cDNA. We religated the plasmid and designatedit pJOD-rsT4 delta E. Then we digested pJOD-rsT4 delta E with NheI andNotI and inserted two DNA fragments: first, an NheI-HindIII linkercontaining a 5′ mRNA splice site and second, a DNA fragment encoding theconstant region of an IgG heavy chain gene. We obtained these fragmentsas follows.

We synthesized an NheI-HindIII linker having the following sequence:                5′ splice 5′ CTA GCT TTC CAA GGT GAG TCC TA      3′3′      GA AAG GTT CCA CTC AGG ATT CGA 5′

The DNA sequence of an IgG heavy chain gene is described in Ellison etal. (1982). We isolated a fragment of this gene from an EMBL3 humangenomic library (Example VIII) using an oligonucleotide probe. Wedigested the fragment with HindIII and NotI and isolated the fragmentwhich included the constant heavy domains and the associated introns.

We ligated these two fragments into PJOD-rsT4 delta E and called theresulting plasmid pAB53. We digested pAB53 with XhoI and NheI to deletethe rsT4 coding region. We inserted in its place the XhoI-NheI fragmentencoding VCAM1 domains 1-3. We called this plasmid VCAM1-IgG₁.

An rsVCAM1/IgG fusion protein is expressed using this plasmid. Theplasmid is transfected into CHO cells for stable expression. Aftertranscription of this gene, the mRNA is spliced to remove the intronsand upon translation, the cell produces rsVCAM-IgG fusion protein.

EXAMPLE XVII Inhibiting VCAM1 Expression with an Antisense Nucleic Acid

We describe here an antisense nucleic acid against VCAM1 and a methodfor testing its ability to inhibit VCAM1 expression in induced HUVECs.An effective nucleic acid sequence for an antisense nucleic acid is onethat is complementary to the coding region of the mRNA and, moreparticularly, to either the initiation codon, AUG, or the splice sites.(Marcus-Sekura, 1988.) Also, oligomers of about 15 nucleotides are mostpreferred. Thus, an effective antisense nucleic acid against VCAM1 is anoligomer with the DNA sequence 5′ CCC AGG CAT TTT AAG. This would bindto nucleotides 94-108 of FIG. 3 (CAT is the antisense initiation codon.)This DNA sequence is synthesized, for example, by an automated DNAsynthesizer.

The ability of this antisense nucleic acid to inhibit VCAM1 expressionis tested as follows. HUVECs are grown to confluence as in Example Vexcept that the serum used for cell growth would be heat inactivated or30 min. at 60° to inactivate nucleases. Cells are preincubated with theoligomers at concentrations between 10 μM and 100 μM, most preferablythe highest concentration having no effect on cell viability, for fourto forty-eight hours. These ranges are required for effectiveinhibition. (Marcus-Sekura, 1988; Becker et al., 1989.) The HUVECs arethen treated with 10 ng/ml TNF to induce VCAM1. About four hours laterthe presence of VCAM1 on the surface of the cells is tested by theadhesion assay.

EXAMPLE XVIII A Hammerhead Ribozyme Which Recognizes VCAM1 mRNA

A hammerhead-type ribozyme which recognizes VCAM1 mRNA is preparedaccording to the rules of Haselhoff and Gerlach (1988) as follows.First, a cleavage site on the target mRNA is identified. Hammerheadribozymes cleave after the sequence 5′ GUX, where X is any nucleotide.The first instance of this sequence in the coding region of VCAM1 mRNAis the sixth codon: 5′ AUG CCU GGG AAG AUG GUC GUG AUC CUU. Anappropriate recognition sequence includes about six nucleotides of the5′ and 3′ regions flanking the cleavage site. An eighteen-baserecognition sequence which contains the cleavage site is 5′ AAG AUG GUCGUG AUC CUU.

Then, one designs an RNA sequence for the ribozyme containing therecognition sequence and a sequence for the catalytic “hammerhead.” Sucha sequence is 5′ AAG GAU CAC [CUGAUGAGUCCGUGAGGACGAA] AC CAU CUU. Thesequence in brackets generates the catalytic “hammerhead” and the 5′ and3′ flanking sequences are complementary to and bind to the recognitionsequence. In a similar way, one can also design shorter recognitionsequences or those for other cleavage sites in VCAM1 mRNA or the otherELAM or ELAM ligand mRNAs.

EXAMPLE XIX Anti-Idiotypic Antibodies Recognizing ELAM1 Ligands

We have prepared anti-idiotypic antibodies against anti-ELAM1 antibodiesthat bind to the ligand of ELAM1 on HL-60 cells. We immunized rabbitswith protein-A-purified CDB.BB11.BC6 monoclonal (Example V) emulsified1:1 in complete Freund's adjuvant. Twenty-six days after immunization webled the rabbits and analyzed the anti-sera for specific antibodiesusing FACS. We incubated the antibody preparation with either HL-60cells, which express a ligand for ELAM1, or RAMOS cells, which do not.We found that this antibody preparation specifically bound to the HL-60cells and not to the RAMOS cells, indicating that it containedantibodies that recognize the ELAM1 ligand. Control anti-serum did notreact with either cell line.

EXAMPLE XX Evidence of a New ELAM

The binding of U937 cells (which are monocyte-like) to induced HUVECs isnot blocked by specific Moabs to the ELAM1, VCAM1, and/or ICAM1pathways. U937 binding is blocked, however, by a monoclonal antibody toCD29, the β₁ integrin subunit. This leads us to postulate the existenceof a new adhesion molecule on HUVECs that interacts with leukocytes viaa β₁ integrin. The new molecule is induced with a time-course similar toVCAM1, remaining at maximal levels 48 hours after induction. We havegenerated a subtracted library from 48-hour TNF-treated HUVECS, usingthe methods previously described for the 2.5-hour IL-1 induced HUVECsubtracted sublibrary. We are attempting to clone the new molecule usingthe direct expression protocol described previously.

While we have described herein a number of embodiments of thisinvention, it is apparent that one of skill in the art could alter ourprocedures to provide other embodiments that utilize the processes andcompositions of this invention. Therefore, one will appreciate that thescope of this invention is to be defined by the claims appended heretorather than by the specific embodiments that we have presented by way ofexample.

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1. An isolated antibody that specifically binds to VCAM1. 